[Research progress of co-culture system for constructing vascularized tissue engineered bone].

Science.gov (United States)

Fu, Weili; Xiang, Zhou

2014-02-01

To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

International Nuclear Information System (INIS)

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

1989-01-01

Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

International Nuclear Information System (INIS)

Burger, E.H.; Van der Meer, J.W.; van de Gevel, J.S.; Gribnau, J.C.; Thesingh, G.W.; van Furth, R.

1982-01-01

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45 Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes

Treatment of refractory undifferentiated acute myelogenous leukemia with all-trans-retinoic acid.

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Griggs, J J; Henley, S E; Rowe, J M

1994-02-01

A patient is described with undifferentiated acute myeloblastic leukemia refractory to two courses of daunorubicin and cytosine arabinoside. Because some the myeloblasts developed morphologic features of promyelocytes, the patient was treated with all-trans-retinoic acid (ATRA) in an attempt to promote maturation. Cytogenetic studies and sensitive molecular analysis did not reveal any abnormality classically associated with acute promyelocytic leukemia. Serial bone marrow biopsies demonstrated myeloid maturation, and the patient uneventfully went into a sustained complete remission. A review of the literature confirms this to be an apparently hitherto undescribed response to ATRA that may have therapeutic implications in similar patients.

Promoted neuronal differentiation after activation of alpha4/beta2 nicotinic acetylcholine receptors in undifferentiated neural progenitors.

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Takeshi Takarada

Full Text Available BACKGROUND: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains. METHODOLOGY/PRINCIPAL FINDINGS: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4β2 nAChR subtype antagonists dihydro-β-erythroidine and 4-(5-ethoxy-3-pyridinyl-N-methyl-(3E-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

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Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

Undifferentiated Gender Role Orientation, Drinking Motives, and Increased Alcohol Use in Men and Women.

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Fugitt, Jessica L; Ham, Lindsay S; Bridges, Ana J

2017-05-12

Alcohol misuse has historically affected men more than women. However, the differences in drinking behaviors across sex have steadily decreased over time and accumulating research suggests that gender role orientation, or culturally scripted gender-specific characteristics, and negative reinforcement drinking motives may better explain risk for alcohol use and related problems than sex. The current study tested a mediational model of the undifferentiated orientation (low masculinity and low femininity), an oft neglected orientation despite evidence that it could carry much weight in drinking behaviors, versus the other three gender role orientations, coping and conformity drinking motives, and hazardous alcohol use. Participants were 426 current drinkers over age 21 (41% men; 77.8% Caucasian; M age = 34.5, range = 21-73) residing across the United States who completed an online survey. Structural equation modeling analyses suggested that individuals with an undifferentiated orientation (n = 99), compared to masculine (high masculinity, low femininity; n = 102), feminine (high femininity, low masculinity; n = 113), or androgynous (high masculinity, high femininity; n = 112) orientations, reported higher coping drinking motives, which were positively associated with levels of hazardous alcohol use. Although analyses suggested that undifferentiated individuals reported drinking for conformity motives more often than masculine and androgynous individuals, conformity motives were not associated with increased use. Conclusions/Importance: An undifferentiated gender role orientation may contribute a unique risk for alcohol use and related problems by increasing frequency of drinking to cope, a motive specifically associated with hazardous use trajectories.

Can magnetic resonance imaging differentiate undifferentiated arthritis?

DEFF Research Database (Denmark)

Østergaard, Mikkel; Duer, Anne; Hørslev-Petersen, K

2005-01-01

A high sensitivity for the detection of inflammatory and destructive changes in inflammatory joint diseases makes magnetic resonance imaging potentially useful for assigning specific diagnoses, such as rheumatoid arthritis and psoriatic arthritis in arthritides, that remain undifferentiated after...... conventional clinical, biochemical and radiographic examinations. With recent data as the starting point, the present paper describes the current knowledge on magnetic resonance imaging in the differential diagnosis of undifferentiated arthritis....

Porous PEOT/PBT scaffolds for bone tissue engineering: preparation, characterization, and in vitro bone marrow cell culturing

NARCIS (Netherlands)

Claase, M.B.; Grijpma, Dirk W.; Mendes, S.C.; Mendes, Sandra C.; de Bruijn, Joost Dick; Feijen, Jan

2003-01-01

The preparation, characterization, and in vitro bone marrow cell culturing on porous PEOT/PBT copolymer scaffolds are described. These scaffolds are meant for use in bone tissue engineering. Previous research has shown that PEOT/PBT copolymers showed in vivo degradation, calcification, and bone

Bone culture research

Science.gov (United States)

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.

X-radiation-induced differentiation of xenotransplanted human undifferentiated rhabdomyosarcoma

International Nuclear Information System (INIS)

Takizawa, T.; Matsui, T.; Maeda, Y.

1989-01-01

A serially xenotransplantable strain of undifferentiated embryonal rhabdomyosarcoma originating from the nasal cavity of a 42-year-old woman has been established in our laboratory. After radiotherapy for the tumor donor, distinct rhabdomyoblastic differentiation of the undifferentiated sarcoma cells appeared in the primary lesion, and it is a reasonable assumption that X-irradiation has a certain potentiality to induce morphologic differentiation of tumor cells. To study this possibility, tissue fragments of undifferentiated embryonal rhabdomyosarcoma that had grown to more than 10 mm after being transplanted to nude mice were selectively irradiated in situ. The degree of rhabdomyoblastic differentiation according to radiation dose was evaluated by light and electron microscopy and by immunostainability for myoglobin, creatine phosphokinase-MM, and desmin. Distinct morphologic differentiation of undifferentiated sarcoma cells could be induced by repeated X-irradiations at several-week intervals

Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells.

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Garba, Abubakar; Acar, Delphine D; Roukaerts, Inge D M; Desmarets, Lowiese M B; Devriendt, Bert; Nauwynck, Hans J

2017-09-01

Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell

A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

International Nuclear Information System (INIS)

Liu Yanxia; Song Zhihua; Zhao Yang; Qin Han; Cai Jun; Zhang Hong; Yu Tianxin; Jiang Siming; Wang Guangwen; Ding Mingxiao; Deng Hongkui

2006-01-01

Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs

Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

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Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

2009-11-01

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

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Takizawa, Naoki; Okubo, Naoto; Kamo, Masaharu; Chosa, Naoyuki; Mikami, Toshinari; Suzuki, Keita; Yokota, Seiji; Ibi, Miho; Ohtsuka, Masato; Taira, Masayuki; Yaegashi, Takashi; Ishisaki, Akira; Kyakumoto, Seiko

2017-09-15

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Entomophagy and coprophagy in undifferentiated schizophrenia.

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Lingeswaran, Anand; Vijayakumar, Vinayak; Dinesh, John

2009-01-01

Coprophagia or the ingestion of feces, considered to be a variant of pica, has been associated with medical disorders like seizure disorders, cerebral atrophy, and tumors and with psychiatric disorders like mental retardation, alcoholism, depression, obsessive compulsive disorder, schizophrenia, schizoaffective disorder, fetishes, delirium, and dementia. But entomophagy or the practice of eating live or dead insects as food by humans has only been reported as part of eating habits by some cultures in the world and not in association with any medical or neuropsychiatric disorders. Till date, there is no report in medical literature of entomophagy as an association with any neuropsychiatric or medical illnesses. Coprophagy and entomophagy has not been together reported as well. We describe the first ever case report of a 19-year- old male patient diagnosed with undifferentiated schizophrenia and associated with both entomophagy and coprophagy. His schizophrenic symptoms, the entomophagic, coprophagic behaviors improved with olanzapine therapy. Entomophagy and coprophagy, two very unusual human behaviors, can be seen in association with schizophrenia.

Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

DEFF Research Database (Denmark)

Haack-Sørensen, Mandana; Hansen, Susanne Kofoed; Hansen, Louise

2013-01-01

BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit...... differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which...... persists independent of cell density can be used for clinical therapies....

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

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

2015-01-01

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

Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture.

OpenAIRE

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

1987-01-01

The cardiotonic agent amrinone inhibits bone resorption in vitro. Milrinone, an amrinone analog, is a more potent cardiotonic agent with lower toxicity. In contrast to amrinone, milrinone stimulated resorption in cultures of neonatal mouse calvaria and fetal rat limb bones. Threshold doses were 0.1 microM in calvaria and 0.1 mM in limb bones; maximal stimulation occurred in calvaria at 0.1 mM. Maximal responses to milrinone and parathyroid hormone were comparable. Milrinone concentrations bel...

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

International Nuclear Information System (INIS)

Bhattacharyya, M.H.; Whelton, B.D.; Stern, P.H.; Peterson, D.P.

1988-01-01

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 45 Ca release from 45 Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with 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

Clinical and Endoscopic Features of Undifferentiated Gastric Cancer in Patients with Severe Atrophic Gastritis.

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Kishino, Maiko; Nakamura, Shinichi; Shiratori, Keiko

2016-01-01

Differentiated gastric cancer generally develops in the atrophic gastric mucosa, although undifferentiated cancer is sometimes encountered in patients with severe atrophic gastritis. We characterized the endoscopic features of undifferentiated gastric cancer in patients with severe atrophic gastritis. Stage IA early gastric cancer was diagnosed in 501 patients who were admitted to our hospital between April 2003 and March 2012. The endoscopic and pathological findings were compared among 29 patients with undifferentiated cancer and severe atrophic gastritis, 104 patients with undifferentiated cancer and mild/moderate atrophic gastritis and 223 patients with well-differentiated cancer and severe atrophic gastritis. Endoscopic atrophic gastritis was classified according to the Kimura-Takemoto classification as no gastritis, C-1 and C-2 (mild), C-3 and O-1 (moderate) or O-2 and O-3 (severe). The tumors were larger and showed deeper mural invasion in the patients with undifferentiated cancer and severe atrophic gastritis than in those with well-differentiated cancer and severe gastritis or undifferentiated cancer and mild/moderate gastritis. On endoscopy, undifferentiated cancer associated with severe gastritis was often red in color. It is often difficult to diagnose early undifferentiated gastric cancer, especially in patients with severe atrophic gastritis. The present study characterized the important endoscopic features of such tumors.

In vitro interactions of lymphocytes and cultured cells from beagles with plutonium-induced bone tumors

International Nuclear Information System (INIS)

Frazier, M.E.; Lund, J.E.; Busch, R.H.

1976-01-01

Cell cultures have been prepared from lung and bone tumors arising in beagle dogs following exposure to inhaled plutonium. Evaluation of the cultured cells by commonly applied criteria (i.e., cell morphology, lack of contact inhibitory mechanisms, cloning efficiency, growth in soft agar, and tumor production in vivo) indicated that tumor cells were being grown in culture. Blood leukocytes and peripheral lymphocytes from beagle dogs were tested for cytotoxic effects against several cell cultures. Lymphocytes from normal dogs or dogs with unrelated tumors would not kill the bone tumor cells unless monocytes (macrophage) were present, in which case the leukocyte preparation was capable of mounting de novo cytotoxic immune reactions after 3 to 5 days in culture. In contrast, the dogs with plutonium-induced bone tumors had circulating lymphocytes that appeared to have undergone presensitization to bone-tumor-distinctive antigens in vivo. Consequently these lymphocytes interacted with cultured cells promptly after encounter in vitro

Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system.

Directory of Open Access Journals (Sweden)

Lin Wang

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

Intensive combined modality therapy of small round cell and undifferentiated sarcomas in children and young adults

International Nuclear Information System (INIS)

Bader, J.L.; Dewan, R.; Watkins, E.; Kinsella, T.J.; Glatstein, E.; STeinberg, S.M.

1989-01-01

Seventy-five patients (ages 4-35 years) with the following small round cell tumors and undifferentiated sarcoma were treated at the National Cancer Institute: Ewing's sarcome (n=32), peripheral neuroepithelioma (n=14), rhabdomyosarcoma (n=24), undifferentiated sarcoma (n=5). Most patients had poor prognostic features including 36 (48%) with metastatic disease, and 42 (56%) with central (truncal) tumors (22 in the pelvis). Treatment included 5 cycles of intensive induction chemotherapy with vincristine, cyclophosphamide and adriamycin, plus aggressive local radiation therapy using simulation and computerized treatment planning for all patients. Thereafter, complete clinical responses were consolidated with intensive chemotherapy, total body irradiation and autologous bone marrow transplantation. There were three local only failures, 10 local plus distant failures, 36 distant only failures, 3 treatment-related deaths, and one intercurrent death. Overall actuarial survival and event-free survival at 4 years are 49 and 29%, respectively. Actuarial freedom from local progression was seen in 74% of patients at 4 years, quite remarkable considering the bulk and location of most of these tumors. Without aggressive surgery, many of these high risk patients had satisfactory outcomes, but better systemic treatments are still needed.(author). 44 refs.; 8 figs.; 6 tabs

Incidence of trypanosomes in the Canada goose as revealed by bone marrow culture

Science.gov (United States)

Diamond, L.S.; Herman, C.M.

1954-01-01

1. Techniques are described for the cultural isolation of trypanosomes from avian bone marrow obtained from living birds or at autopsy. A new medium SNB-9 (saline-neopeptone-blood) is described. In addition to being a good medium for growing avian trypanosomes, it is excellent for growing trypanosomes of amphibians and mammals. 2. Evidence is presented demonstrating the superiority of (a) cultures over stained smears for detecting the presence of trypanosomes in the Canada goose, and (b) bone marrow over heart blood of this species as a source of trypanosomes for culture. 3. In April 1952, from cultures of bone marrow collected at autopsy it was demonstrated that trypanosome infection occurred in 33 (40.2%) of 82 Canada geese from the Pea Island National Wildlife Refuge. On February 17, 1953, cultures of bone marrow obtained from living birds revealed presence of trypanosomes in 12 (20.7%) of 58 geese from the same refuge. On February 26, 1953, by employing the latter method, 9 (20.4%) of 44 geese from Blackwater National Wildlife Refuge were shown to harbor the parasites. In another survey ninety-two geese from seven national wildlife refuges subjected to the biopsy technique showed evidence of infection in 13 (14.1 %) birds and indicated that trypanosome infection is widely distributed in this host.

Isolation, culture expansion and characterization of canine bone marrow derived mesenchymal stem cells

Directory of Open Access Journals (Sweden)

D Kazemi

2016-07-01

Full Text Available The purpose of the present study was to isolate, culture expand and characterize canine bone marrow derived mesenchymal stem cells. Bone marrow aspirates of 15 adult male dogs were collected to this end and their mononuclear cells isolated by centrifugation and cultured in standard media. The adherent cells were isolated and their mesenchymal origin was confirmed at 3rd passage by cellular morphology, expression of surface antigens and differentiation to osteogenic and adipogenic lineage. After 4 days, spindle shaped fibroblast like cells which were apparently bone marrow derived mesenchymal stem cells appeared in culture medium and their numbers increased over time. The cells reached 3rd passage with over 75% confluent after a mean of 22.89±5.75 days. Flow cytometric analysis revealed that the cells negatively expressed CD34 and CD45 antigens while positively expressing CD44 and CD105 antigens. Differentiation into osteogenic and adipogenic lineage had taken place after one month culture in induction medium. VDR, COL1A1, BGLAP and SPARC gene expression indicated that mesenchymal stem cells isolated from canine bone marrow had differentiated into osteogenic lineage. These findings can form the basis of any forthcoming clinical studies involving the use of canine mesenchymal stem cells particularly in the field of bone and cartilage regeneration.

An in vitro 3D bone metastasis model by using a human bone tissue culture and human sex-related cancer cells.

Science.gov (United States)

Salamanna, Francesca; Borsari, Veronica; Brogini, Silvia; Giavaresi, Gianluca; Parrilli, Annapaola; Cepollaro, Simona; Cadossi, Matteo; Martini, Lucia; Mazzotti, Antonio; Fini, Milena

2016-11-22

One of the main limitations, when studying cancer-bone metastasis, is the complex nature of the native bone environment and the lack of reliable, simple, inexpensive models that closely mimic the biological processes occurring in patients and allowing the correct translation of results. To enhance the understanding of the mechanisms underlying human bone metastases and in order to find new therapies, we developed an in vitro three-dimensional (3D) cancer-bone metastasis model by culturing human breast or prostate cancer cells with human bone tissue isolated from female and male patients, respectively. Bone tissue discarded from total hip replacement surgery was cultured in a rolling apparatus system in a normoxic or hypoxic environment. Gene expression profile, protein levels, histological, immunohistochemical and four-dimensional (4D) micro-CT analyses showed a noticeable specificity of breast and prostate cancer cells for bone colonization and ingrowth, thus highlighting the species-specific and sex-specific osteotropism and the need to widen the current knowledge on cancer-bone metastasis spread in human bone tissues. The results of this study support the application of this model in preclinical studies on bone metastases and also follow the 3R principles, the guiding principles, aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes.

Stimulation of the proliferation of hemopoietic stem cells in irradiated bone marrow cell culture

International Nuclear Information System (INIS)

Mori, K.J.; Izumi, H.; Seto, A.

1981-01-01

Long-term hemopoiesis was established in bone marrow cell culture in vitro. This culture was shown to support the recovery proliferation of hemopoietic stem cells completely in vitro after irradiation. Hemopoietic stem cells were stimulated into proliferation in culture when normal bone marrow cells were overlayed on top of the irradiated adherent cell colonies. These results indicate that proliferation and differentiation of hemopoietic stem cells in vitro are also supported by stromahemopoietic cell interactions

Uptake of a fluorinated bisphosphonate by cultured bones

International Nuclear Information System (INIS)

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

Primary renal undifferentiated sarcoma as an infiltrative mass in a 12 year old boy

International Nuclear Information System (INIS)

Kim, Yong Hee; Kim, Myung Joon; Lee, Mi Jung; Kim, Se Hwa

2015-01-01

Undifferentiated sarcomas are rare tumors not classified into any sarcoma subtype. Due to their rarity, imaging findings of undifferentiated sarcomas are poorly characterized. The purpose of this report was to present imaging findings of a pathologically confirmed undifferentiated sarcoma originated from the left kidney of a 12-year-old boy. The mass was infiltrative involving the renal pelvis. It mimicked massive hilar lymphadenopathy with a preserved renal contour visible by both ultrasonography and CT. Renal vein thrombosis was also observed. Although undifferentiated sarcomas are rare, they should be considered in differential diagnosis of infiltrative renal masses with renal pelvis invasion in children

Primary renal undifferentiated sarcoma as an infiltrative mass in a 12 year old boy

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Kim, Yong Hee; Kim, Myung Joon; Lee, Mi Jung [Dept. of Radiology and Research Institute of Radiological Science, Severance Children' s Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Se Hwa [Dept. of Pathology, Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of)

2015-09-15

Undifferentiated sarcomas are rare tumors not classified into any sarcoma subtype. Due to their rarity, imaging findings of undifferentiated sarcomas are poorly characterized. The purpose of this report was to present imaging findings of a pathologically confirmed undifferentiated sarcoma originated from the left kidney of a 12-year-old boy. The mass was infiltrative involving the renal pelvis. It mimicked massive hilar lymphadenopathy with a preserved renal contour visible by both ultrasonography and CT. Renal vein thrombosis was also observed. Although undifferentiated sarcomas are rare, they should be considered in differential diagnosis of infiltrative renal masses with renal pelvis invasion in children.

Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.

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Liu, Yuchun; Chan, Jerry K Y; Teoh, Swee-Hin

2015-02-01

Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.

Radiation therapy for primary undifferentiated carcinoma of the esophagus

International Nuclear Information System (INIS)

Ohno, Tatsuya; Yamakawa, Michitaka; Shiojima, Kazumi; Hasegawa, Masatoshi; Akimoto, Tetsuo; Nakayama, Yuko; Kitamoto, Yoshizumi; Mitsuhashi, Norio; Niibe, Hideo

1996-01-01

Eight patients with undifferentiated carcinoma of the esophagus were treated by radiation therapy. Loco-regional control was easily achieved by radiation therapy alone and no loco-regional recurrence was observed for six patients treated with total dose of more than 30 Gy. However four patients developed distant metastases and died of tumor. Median survival was 3.5 months with a range of 0 to 48 months. Only one patient is alive with no evidence of tumor for 48 months. Combination chemotherapy should be recommended for primary undifferentiated carcinoma of the esophagus because of having a high incidence of distant metastases. (author)

Proliferation differentiation and therapeutic effect of short-term cultured murine bone marrow cells

International Nuclear Information System (INIS)

Zhao Zekun; Cong Jianbo

1986-01-01

Murine bone marrow cells were cultured in conditioned medium of muscle. After 24 hours of culture, both adherent and suspended cells appeared in the culture. The adherent cells mainly consisted of macrophages and the suspended cells were predominantly granulocytes. After 6 days, the total number of nucleated cells and CFU-C in the culture increased about 400% and 600% respectively, but CFU-S reduced to 21% approximately. Lymphocytes persisted only for 4 days. The stem cells (CFU-S) from 6-day culture were injected into the lethally irradiated syngenic mice. The 30 day survival rate of the treated mice was 89% whereas that of the controls was only 7%. The bone marrow cells in 2/8 of recipients sacrificed at 30 or 60 days were of donor type and 6/8 of the recipients were chimeras

Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture.

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

1987-01-01

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

Multiple bone metastasis of medulloblastoma; a case report

International Nuclear Information System (INIS)

Oh, Jae Cheon; Lee, Seoung Ro; Kim, Yong Soo; Park, Dong Woo; Joo, Kyung Bin; Hahm, Chang Kok

1996-01-01

Medulloblastoma is one of the most undifferentiated primitive neuroectodermal tumors and represents about 30% of all posterior fossa tumors in children. Disseminated medulloblastoma, mainly involving cerebral surfaces, ventricles and the subarachnoid space can, in 50% of patients, be identified on intial imaging studies. One third of these lesions metastasize to an extracranial sity, primarily to bone. Osseous metastases, which occur mainly after craniectomy are typically lytic, but osteoblastic lesions also may occur. We experienced the case of a 14 year-old female patient with multiple bone metastases of medulloblastoma after craniectomy. Bone metastatic lesions were present in the right femur and thoracic spine and were osteoblastic or osteolytic

Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

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María Berdasco

Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

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Ranera Beatriz

2012-08-01

Full Text Available Abstract Background Mesenchymal stem cells (MSCs derived from bone marrow (BM-MSCs and adipose tissue (AT-MSCs are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2. This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. Results At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. Conclusions Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

Transcription profiling by array of the response of Arabidopsis cultivar Columbia etiolated seedlings and undifferentiated tissue culture cells to the spaceflight environment

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National Aeronautics and Space Administration — We address a key baseline question of whether gene expression changes are induced by the orbital environment and then we ask whether undifferentiated cells cells...

Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

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Akahane, M; Shimizu, T; Kira, T; Onishi, T; Uchihara, Y; Imamura, T; Tanaka, Y

2016-11-01

To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1. © 2016 Akahane et al.

Undifferentiated carcinoma of the esophagus: a clinicopathological study of 16 cases☆

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Singhi, Aatur D.; Seethala, Raja R.; Nason, Katie; Foxwell, Tyler J.; Roche, Robyn L.; McGrath, Kevin M.; Levy, Ryan M.; Luketich, James D.; Davison, Jon M.

2015-01-01

Summary Undifferentiated carcinoma of the esophagus is a rare histologic variant of esophageal carcinoma. Using criteria based on studies of undifferentiated carcinomas arising at other sites, we have collected 16 cases of resected esophageal undifferentiated carcinomas. Patients ranged in age from 39 to 84 years (mean, 65.5 years) and were predominantly male (94%). The tumors were characterized by an expansile growth pattern of neoplastic cells organized in solid sheets and without significant glandular, squamous, or neuroendocrine differentiation. The neoplastic cells had a syncytial-like appearance, little intervening stroma, and patchy tumor necrosis. In a subset of cases, the tumor cells adopted a sarcomatoid (n = 2), rhabdoid (n = 1), or minor component (esophagus. Consistent with the epithelial nature of these neoplasms, cytokeratin positivity was identified in all cases. In addition, SALL4 expression was present in 8 (67%) of 12 cases. Follow-up information was available for 15 (94%) of 16 patients, all of whom were deceased. Survival after surgery ranged from 1 to 50 months (mean, 11.9 months). Before death, 67% patients had documented locoregional recurrence and/or distant organ metastases. In summary, esophageal undifferentiated carcinomas are aggressive neoplasms and associated with a high incidence of recurrence and/or metastases and a dismal prognosis. PMID:25582499

Noun-Verb Ambiguity in Chronic Undifferentiated Schizophrenia

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Goldfarb, Robert; Bekker, Natalie

2009-01-01

This study investigated noun-verb retrieval patterns of 30 adults with chronic undifferentiated schizophrenia and 67 typical adults, to determine if schizophrenia affected nouns (associated with temporal lobe function) differently from verbs (associated with frontal lobe function). Stimuli were homophonic homographic homonyms, balanced according…

Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

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Tsao, C J; Cheng, T Y; Chang, S L; Su, W J; Tseng, J Y

1992-05-01

We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

Role of bone marrow-derived stem cells, renal progenitor cells and ...

African Journals Online (AJOL)

It remains the leading cause of late allograft loss. Bone marrow derived stem cells are undifferentiated cells typically characterized by their capacity for self renewal, ability to give rise to multiple differentiated cellular population, including hematopoietic (HSCs) and mesenchymal stem cells (MSCs). Characterization of HSCs ...

Characteristics of monolayer culture of bone marrow cells of rats bearing 239Pu-induced osteosarcoma

International Nuclear Information System (INIS)

Bukhtoyarova, Z.M.; Lemberg, V.K.

1984-01-01

The report is concerned with a monolayer culture of bone marrow cells of rats in which optimal blastogenic dose (92.5 kBq/kg) induced osteosarcoma. The cell culture showed an enhanced rate of fibroblast-like cell proliferation (increased number of mitoses and symplasts and larger colonies of cells), apparent signs of radiation in ury (pathologic mitoses, chromosome aberrations and gaps) as well as an increase in ploidy. Diffusion chamber measurements demonstrated osteogenic precursor-cells in osteosarcoma-bearing rats to be highly capable of bone formation. This relatively high ability seems to occur outside bone marrow as well

Prevascularization of 3D printed bone scaffolds by bioactive hydrogels and cell co-culture.

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Kuss, Mitchell A; Wu, Shaohua; Wang, Ying; Untrauer, Jason B; Li, Wenlong; Lim, Jung Yul; Duan, Bin

2017-09-13

Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

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Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

2011-08-01

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Possibility of Undifferentiated Human Thigh Adipose Stem Cells Differentiating into Functional Hepatocytes

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Jong Hoon Lee

2012-11-01

Full Text Available BackgroundThis study aimed to investigate the possibility of isolating mesenchymal stem cells (MSCs from human thigh adipose tissue and the ability of human thigh adipose stem cells (HTASCs to differentiate into hepatocytes.MethodsThe adipose-derived stem cells (ADSCs were isolated from thigh adipose tissue. Growth factors, cytokines, and hormones were added to the collagen coated dishes to induce the undifferentiated HTASCs to differentiate into hepatocyte-like cells. To confirm the experimental results, the expression of hepatocyte-specific markers on undifferentiated and differentiated HTASCs was analyzed using reverse transcription polymerase chain reaction and immunocytochemical staining. Differentiation efficiency was evaluated using functional tests such as periodic acid schiff (PAS staining and detection of the albumin secretion level using enzyme-linked immunosorbent assay (ELISA.ResultsThe majority of the undifferentiated HTASCs were changed into a more polygonal shape showing tight interactions between the cells. The differentiated HTASCs up-regulated mRNA of hepatocyte markers. Immunocytochemical analysis showed that they were intensely stained with anti-albumin antibody compared with undifferentiated HTASCs. PAS staining showed that HTASCs submitted to the hepatocyte differentiation protocol were able to more specifically store glycogen than undifferentiated HTASCs, displaying a purple color in the cytoplasm of the differentiated HTASCs. ELISA analyses showed that differentiated HTASCs could secrete albumin, which is one of the hepatocyte markers.ConclusionsMSCs were islolated from human thigh adipose tissue differentiate to heapatocytes. The source of ADSCs is not only abundant abdominal adipose tissue, but also thigh adipose tissue for cell therapy in liver regeneration and tissue regeneration.

Dedifferentiated giant-cell tumor of bone with an undifferentiated round cell mesenchymal component

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Eréndira G. Estrada-Villaseñor

2014-08-01

Full Text Available The dedifferentiated giant-cell tumor of the bone is a very rare variant of the giant-cell tumor (GCT. We report the clinical, radiographic and histological findings of a dedifferentiated GCT in which the dedifferentiated component consisted of small round cells. We also comment on previously reported cases of dedifferentiated GCT, discuss the clinical implications of this dual histology, and analyze the information published about the coexistence of similar genetic abnormalities in GCT and small round cell tumors of the bone.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis.

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Sieh, Shirly; Taubenberger, Anna V; Lehman, Melanie L; Clements, Judith A; Nelson, Colleen C; Hutmacher, Dietmar W

2014-06-01

As microenvironmental factors such as three-dimensionality and cell-matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to

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

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Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)

2012-04-05

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.

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Development of a mechanical testing and loading system for trabecular bone studies for long term culture

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DB Jones

2003-03-01

Full Text Available A highly accurate (�3% mechanical loading and measurement system combined with a trabecular bone diffusion culture-loading chamber has been developed, which provides the ability to study trabecular bone (and possibly cartilage under controlled culture and loading conditions over long periods of time. The loading device has been designed to work in two main modes, either to apply a specific compressive strain to a trabecular bone cylinder or to apply a specific force and measure the resulting deformation. Presently, precisely machined bone cylinders can be loaded at frequencies between 0.1 Hz to 50 Hz and amplitudes over 7,000�e. The system allows accurate measurement of many mechanical properties of the tissue in real time, including visco-elastic properties. This paper describes the technical components, reproducibility, precision, and the calibration procedures of the loading system. Data on long term culture and mechanical responses to different loading patterns will be published separately.

Undifferentiated pleomorphic sarcoma with osteoclast-like giant cells of the female breast

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Balbi Giancarlo

2013-01-01

Full Text Available Abstract The authors describe a case of undifferentiated pleomorphic sarcoma of the breast occurring in a 50-year-old woman who presented with a palpable mass in her right breast. She first noticed the mass one month previously. Core needle biopsy showed connective tissue including epithelioid and spindle cells. The patient underwent total mastectomy without axillary lymph node dissection. Based on examination of the excised tumor, the initial pathologic diagnosis was atypical spindle-shaped and ovoid cells with uncertain malignant potential. Histological findings with immunomarkers led to the final diagnosis of undifferentiated pleomorphic sarcoma. This case highlights a rare and interesting variant of primary breast sarcoma and the important role of immunohistochemistry in defining histological type and differential diagnosis. Hence, undifferentiated pleomorphic sarcoma has been a diagnosis of exclusion performed through sampling and critical use of ancillary diagnostic techniques.

Priming 3D cultures of human mesenchymal stromal cells toward cartilage formation via developmental pathways.

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Centola, Matteo; Tonnarelli, Beatrice; Schären, Stefan; Glaser, Nicolas; Barbero, Andrea; Martin, Ivan

2013-11-01

The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.

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

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María D. Cuenca-López

2014-12-01

Full Text Available 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

[Nailfold capillaroscopy in the evaluation of Raynaud's phenomenon and undifferentiated connective tissue disease].

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Cortes, Sara; Clemente-Coelho, Paulo

2008-01-01

Microvascular abnormalities involved in the pathogenic mechanism of several connective tissue disorders can be detected by nailfold capillaroscopy. Evaluation of the interest of nailfold capillaroscopy results in patients with Raynaud s phenomenon or undifferentiated connective tissue disease and their correlation with diagnostic and therapeutical evolution. Selection of capillaroscopic and laboratory results of patients with the diagnosis of Raynaud s phenomenon (without defined connective tissue disease) or undifferentiated connective tissue disease. Evaluation of the present diagnosis and treatment comparing with the ones existed at the time of capillaroscopy performance. 80 patients were enrolled with an age of 51.4+/-14.3 years (mean+/-SD) 78 females (97.5%) with Raynaud s phenomenon and undifferentiated connective tissue disease 27 patients (33.8%); Raynaud s Phenomenon 46 patients (57.5%); undifferentiated connective tissue disease 7 patients (8.7%). The capillaroscopic results were normal 30 patients (37.5%); minor changes tortuosity enlargement 16 patients (20.0%) major changes 34 patients (42.5%) hemorrhages 25 patients (31.3%) megacapillaries 26 patients (32.5%) avascular areas 3 patients (3.8%). The introduction of new treatments after the capillaroscopy occurred in 32 patients (40.0%) and a new diagnosis was done in 39 patients (48.8%). Major changes in capillaroscopy correlated with the change of diagnosis and the introduction of a new treatment (pNailfold capillaroscopy performed in patients with isolated Raynaud s phenomenon or undifferentiated connective tissue disease has a role in the prognostic evaluation related to the possibility of an evolution of the diagnosis or to the need of the introduction of new treatments.

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

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Maeda, Eijiro; Nakagaki, Masashi; Ichikawa, Katsuhisa; Nagayama, Kazuaki; Matsumoto, Takeo

2017-06-01

Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture

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Eijiro Maeda

2017-06-01

Full Text Available Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3–4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen

ZEB1 overexpression associated with E-cadherin and microRNA-200 downregulation is characteristic of undifferentiated endometrial carcinoma.

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Romero-Pérez, Laura; López-García, M Ángeles; Díaz-Martín, Juan; Biscuola, Michele; Castilla, M Ángeles; Tafe, Laura J; Garg, Karuna; Oliva, Esther; Matias-Guiu, Xavier; Soslow, Robert A; Palacios, José

2013-11-01

Undifferentiated endometrial carcinomas are very aggressive high-grade endometrial carcinomas that are frequently under-recognized. This study aimed to analyze the molecular alterations underlying the development of these endometrial carcinomas, focusing on those related to dedifferentiation. We assessed a series of 120 tumors: 57 grade 1 and 2 endometrioid endometrial carcinomas, 15 grade 3 endometrioid endometrial carcinomas, 27 endometrial serous carcinomas, and 21 undifferentiated endometrial carcinomas. We found a high frequency of DNA mismatch repair deficiency (38%) and moderate rate of p53 overexpression (∼33%) in undifferentiated carcinomas. In contrast to the characteristic endometrioid phenotype, there was a dramatic downregulation of E-cadherin expression in the undifferentiated subtype. Quantitative methylation studies dismissed CDH1 promoter hypermethylation as the mechanism responsible for this change in gene expression, while immunohistochemistry revealed that the E-cadherin repressor ZEB1 was frequently overexpressed (62%) in undifferentiated endometrial carcinomas. This finding was accompanied by a sharp downregulation in the expression of the miR-200 family of microRNAs, well-known targets of ZEB1. Furthermore, there was enhanced expression of epithelial-to-mesenchymal transition markers in undifferentiated endometrial carcinomas, such as N-cadherin, cytoplasmic p120, and osteonectin. In addition, HMGA2, a regulator of epithelial-to-mesenchymal transition that is expressed in aggressive endometrial tumors, such as endometrial serous carcinomas and carcinosarcomas, was expressed in >20% of undifferentiated carcinomas. These results suggest that ZEB1 overexpression, associated with E-cadherin and miR-200s downregulation, and the expression of mesenchymal markers might enhance the metastatic potential of undifferentiated endometrial carcinomas, leading to a poor prognosis. In addition, our observations suggest that the immnohistochemical analysis

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

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Gesiane Ribeiro

2013-12-01

Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

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

International Nuclear Information System (INIS)

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

1985-01-01

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 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 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

Bem Sex Role Inventory Undifferentiated Score: A Comparison of Sexual Dysfunction Patients with Sexual Offenders.

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Dwyer, Margretta; And Others

1988-01-01

Examined Bem Sex Role undifferentiated scores on 93 male sex offenders as compared with 50 male sexually dysfunctional patients. Chi-square analyses revealed significant difference: offenders obtained undifferentiated scores more often than did sexual dysfunctional population. Concluded that Bem Sex Role Inventory is useful in identifying sexual…

Role of whole bone marrow, whole bone marrow cultured cells, and mesenchymal stem cells in chronic wound healing.

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Rodriguez-Menocal, Luis; Shareef, Shahjahan; Salgado, Marcela; Shabbir, Arsalan; Van Badiavas, Evangelos

2015-03-13

Recent evidence has shown that bone marrow cells play critical roles during the inflammatory, proliferative and remodeling phases of cutaneous wound healing. Among the bone marrow cells delivered to wounds are stem cells, which can differentiate into multiple tissue-forming cell lineages to effect, healing. Gaining insight into which lineages are most important in accelerating wound healing would be quite valuable in designing therapeutic approaches for difficult to heal wounds. In this report we compared the effect of different bone marrow preparations on established in vitro wound healing assays. The preparations examined were whole bone marrow (WBM), whole bone marrow (long term initiating/hematopoietic based) cultured cells (BMC), and bone marrow derived mesenchymal stem cells (BM-MSC). We also applied these bone marrow preparations in two murine models of radiation induced delayed wound healing to determine which had a greater effect on healing. Angiogenesis assays demonstrated that tube formation was stimulated by both WBM and BMC, with WBM having the greatest effect. Scratch wound assays showed higher fibroblast migration at 24, 48, and 72 hours in presence of WBM as compared to BM-MSC. WBM also appeared to stimulate a greater healing response than BMC and BM-MSC in a radiation induced delayed wound healing animal model. These studies promise to help elucidate the role of stem cells during repair of chronic wounds and reveal which cells present in bone marrow might contribute most to the wound healing process.

Cytokine and immunoglobulin production by PWM-stimulated peripheral and tumor-infiltrating lymphocytes of undifferentiated nasopharyngeal carcinoma (NPC patients

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Bouzouita Kamel

2004-09-01

Full Text Available Abstract Background Undifferentiated Nasopharyngeal Carcinoma (NPC patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL and tumor- infiltrating (TIL lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10 and three immunoglobulin isotypes (IgM, IgG, IgA. Methods Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA. Data were analysed using Student's t-test and probability values below 5% were considered significant. Results The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002, IL-10 (p = 0,020, IgM (p= 0,0003 and IgG (p Conclusion Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC.

DENTAL PULP STEM CELLS AND HUMAN PERIAPICAL CYST MESENCHYMAL STEM CELLS IN BONE TISSUE REGENERATION: COMPARISON OF BASAL AND OSTEOGENIC DIFFERENTIATED GENE EXPRESSION OF A NEWLY DISCOVERED MESENCHYMAL STEM CELL LINEAGE.

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Tatullo, M; Falisi, G; Amantea, M; Rastelli, C; Paduano, F; Marrelli, M

2015-01-01

Bone regeneration is an interesting field of biomedicine. The most recent studies are aimed to achieve a bone regeneration using mesenchymal stem cells (MSCs) taken from more accessible sites: oral and dental tissues have been widely investigated as a rich accessible source of MSCs. Dental Pulp Stem Cells (DPSCs) and human Periapical Cysts Mesenchymal Stem Cells (hPCy-MSCs) represent the new generation MSCs. The aim of this study is to compare the gene expression of these two innovative cell types to highlight the advantages of their use in bone regeneration. The harvesting, culturing and differentiating of cells isolated from dental pulp as well as from periapical cystic tissue were carried out as described in previously published reports. qRT-PCR analyses were performed on osteogenic genes in undifferentiated and osteogenic differentiated cells of DPSC and hPCy-MSC lineage. Real-time RT-PCR data suggested that both DPSCs and hPCy-MSCs cultured in osteogenic media are able to differentiate into osteoblast/odontoblast-like cells: however, some differences indicated that DPSCs seem to be directed more towards dentinogenesis, while hPCy-MSCs seem to be directed more towards osteogenesis.

Review: Rainer Diaz-Bone (2002. Kulturwelt, Diskurs und Lebensstil [Cultural World, Discourse and Life Style

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Johannes Angermüller

2004-01-01

Full Text Available In his work on the discourse of popular music DIAZ-BONE opens BOURDIEU's theory of distinction to a discursive approach. Analyzing the discourse of German heavy metal and techno zines, the author demonstrates the usefulness of a theory of discourse based on the notion of difference and opposition for the sociology of culture. He criticizes BOURDIEU for his reductionist tendency and underlines the relative autonomy of discursive patterns for establishing a system of hierarchical cultural values. In his analysis DIAZ-BONE finds that the discourse about heavy metal music gives, amongst others, a prominent role to "authenticity" and "artisan solidity", whereas the discourse about techno DJs rather highlights technical innovation and cultural networking. URN: urn:nbn:de:0114-fqs040126

Undifferentiated-type gastric adenocarcinoma: prognostic impact of three histological types

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Lee Han

2012-11-01

Full Text Available Abstract Background The prognostic value of the three constituents of undifferentiated-type gastric adenocarcinoma remains unclear. The present study assessed the clinicopathological characteristics and prognosis of undifferentiated-type mucinous adenocarcinoma (uMAC and signet ring cell carcinoma (SRC compared with those of poorly differentiated adenocarcinoma (PDAC. Methods In total, 1,376 patients with undifferentiated-type gastric adenocarcinoma were included, consisting of 1,002 patients diagnosed with PDAC, 54 with uMAC and 320 with SRC. Clinicopathological factors and survival rates were compared among the three histological types. Results Significant differences in the distribution of pathological stages were observed among the groups. Patients with SRC had a significantly better survival rate than those with PDAC or uMAC, in both the all patients including non-curative resected patients and curative-resected groups. In addition, there was significant difference in survival between the PDAC and uMAC groups. Multivariate analysis suggested that age, gender, tumor depth, lymph node metastasis and curability significantly affected survival. Histological type was not an independent prognostic factor. There was no significant difference in the pattern of recurrence among the three groups. Conclusions The uMAC and SRC had worse and favorable prognosis compared with PDCA, respectively. However, there were no differences in survival by pathological stage, thus histological type was not an independent predictor of prognosis.

Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

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Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

2015-11-17

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

Primary undifferentiated sarcoma of the meninges: A case report and comprehensive review of the literature.

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Wapshott, Taylor; Schammel, Christine M G; Schammel, David P; Rezeanu, Luminita; Lynn, Michael

2018-05-21

Sarcomas make up 1% of all cases of adult cancer, with 5-10% of those classified as undifferentiated pleomorphic sarcomas (UPS/PUS) and 0.1-4.3% primary intracranial sarcomas. Intracranial undifferentiated sarcoma is characterized by an earlier age of onset and generally poorer prognosis compared to extracranial undifferentiated sarcomas. Current therapies involve surgical excision with wide margins and radiotherapy, with minimal data available regarding the efficacy of chemotherapy. A 79-year-old man with a history of remote superficial bladder cancer presented with a large frontal scalp lesion. A biopsy was initially attempted by a dermatologist in the outpatient setting, but a follow-up CT scan revealed a skull-eroding, enhancing soft tissue lesion. Neurosurgical treatment revealed an undifferentiated sarcoma. The patient underwent adjuvant radiation therapy of 59.4 Gy fractionated over 45 days following surgery. Follow-up brain MRIs at 1-, 6-, 9-, 12-, 15-, 21-, and 27 months after surgery have not shown any indications of local recurrence or tumor metastasis. Despite the high propensity that undifferentiated sarcomas have for recurrence and metastasis and the patient's advanced age, this patient remains uniquely disease-free. We provide a description of an unusual case and comprehensive literature review of UPS to clarify the hallmarks of the disease, identify the difficulties in diagnosis, and provide a summary of therapies employed in the literature with their corresponding patient outcomes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Postradiation recovery of the bone marrow of man and morphodynamics of the pool of undifferentiated cells

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Suvorova, L.A.; Vyalova, N.A.; Barabanova, A.V.; Gruzdev, G.P.

1981-01-01

Peculiarities of postradiation recovery of bone marrow parenchyma and stroma in persons who have been exposed to uniform gamma irradiation and nonuniform gamma-neutron irradiation at doses of 2-5 and more than 5 Gy are described on the basis of quantitive characteristics of bone marrow trepanates which have been investigated in different periods of acute radiation sickness (from 2 to 43 days). A special attention is paid to the description of the behaviour of clones composed of nondifferentiated cells that appear in bone marrow of the 4th - 6th day of sickness and participate in its repair. The obtained results attest that clone-forming nondifferentiated cells are the basis for hemopoetic parenchyma recovery. The number of trunk hemopoetic cells in this or that area of bone marrow exposed to the irradiation at different doses can be determined with a high degree of probability by the number of clones.

Imaging features of undifferentiated embryonal sarcoma of the liver: a series of 15 children

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Gabor, Flaviu; Franchi-Abella, Stephanie; Pariente, Daniele [Bicetre Hospital, Department of Pediatric Radiology, Le Kremlin-Bicetre (France); Merli, Laura [Bambino Gesu Children' s Hospital, Unit of Hepato-Biliary and Transplant Surgery, Department of Surgery and Transplantation Centre, Rome (Italy); Adamsbaum, Catherine [Bicetre Hospital, Department of Pediatric Radiology, Le Kremlin-Bicetre (France); Paris Sud University, Faculty of Medicine, Le Kremlin-Bicetre (France); Universite Paris-Saclay, LTCI, CNRS, Telecom Paris Tech, Paris (France)

2016-11-15

Undifferentiated embryonal sarcoma of the liver is a rare malignant mesenchymal tumour occurring mostly in children ages 6-10 years. The discrepancy between its solid appearance on US and cystic-like appearance on CT has been described. To study the imaging particularities and similarities among our cases of undifferentiated embryonal sarcoma and to report the errors in initial diagnoses. We conducted a retrospective study of 15 children with undifferentiated embryonal sarcoma diagnosed or referred to our hospital during 1997-2015 and analysed the clinical, biological and imaging data. We identified eight boys and seven girls ages 9 months to 14 years. Ten children presented with abdominal pain. Alpha-fetoprotein was slightly increased in one. Initial US and CT had been performed for all, while additional MRI had been done in two children. Initial CT demonstrated a hypoattenuated mass in all. Rupture was seen in five and intratumoural bleeding in seven children. Tumour volumes reduced during neoadjuvant chemotherapy in 10 children. Undifferentiated embryonal sarcoma might be suggested in a non-secreting unifocal tumour with well-defined borders, fluid-filled spaces on US, hypoattenuation and serpiginous vessels on CT, and if there are signs of internal bleeding or rupture on CT or MRI. (orig.)

Imaging features of undifferentiated embryonal sarcoma of the liver: a series of 15 children

International Nuclear Information System (INIS)

Gabor, Flaviu; Franchi-Abella, Stephanie; Pariente, Daniele; Merli, Laura; Adamsbaum, Catherine

2016-01-01

Undifferentiated embryonal sarcoma of the liver is a rare malignant mesenchymal tumour occurring mostly in children ages 6-10 years. The discrepancy between its solid appearance on US and cystic-like appearance on CT has been described. To study the imaging particularities and similarities among our cases of undifferentiated embryonal sarcoma and to report the errors in initial diagnoses. We conducted a retrospective study of 15 children with undifferentiated embryonal sarcoma diagnosed or referred to our hospital during 1997-2015 and analysed the clinical, biological and imaging data. We identified eight boys and seven girls ages 9 months to 14 years. Ten children presented with abdominal pain. Alpha-fetoprotein was slightly increased in one. Initial US and CT had been performed for all, while additional MRI had been done in two children. Initial CT demonstrated a hypoattenuated mass in all. Rupture was seen in five and intratumoural bleeding in seven children. Tumour volumes reduced during neoadjuvant chemotherapy in 10 children. Undifferentiated embryonal sarcoma might be suggested in a non-secreting unifocal tumour with well-defined borders, fluid-filled spaces on US, hypoattenuation and serpiginous vessels on CT, and if there are signs of internal bleeding or rupture on CT or MRI. (orig.)

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

Science.gov (United States)

Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

2009-04-01

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Osteoclasts secrete non-bone derived signals that induce bone formation

DEFF Research Database (Denmark)

Karsdal, Morten A; Neutzsky-Wulff, Anita V; Dziegiel, Morten Hanefeld

2008-01-01

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption......) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts...

Addition of exogenous cytokines in mixed lymphocyte culture for selecting related donors for bone marrow transplantation

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Jeane Eliete Laguila Visentainer

Full Text Available CONTEXT: Mixed lymphocyte culturing has led to conflicting opinions regarding the selection of donors for bone marrow transplantation. The association between a positive mixed lymphocyte culture and the development of graft-versus-host disease (GVHD is unclear. The use of exogenous cytokines in mixed lymphocyte cultures could be an alternative for increasing the sensitivity of culture tests. OBJECTIVE: To increase the sensitivity of mixed lymphocyte cultures between donor and recipient human leukocyte antigen (HLA identical siblings, using exogenous cytokines, in order to predict post-transplantation GVHD and/or rejection. TYPE OF STUDY: Prospective study. SETTING: Bone Marrow Transplantation Unit, Universidade Estadual de Campinas. PARTICIPANTS: Seventeen patients with hematological malignancies and their respective donors selected for bone marrow transplantation procedures. PROCEDURES: Standard and modified mixed lymphocyte culturing by cytokine supplementation was carried out using donor and recipient cells typed for HLA. MAIN MEASUREMENTS: Autologous and allogenic responses in mixed lymphocyte cultures after the addition of IL-4 or IL-2. RESULTS: In comparison with the standard method, average responses in the modified mixed lymphocyte cultures increased by a factor of 2.0 using IL-4 (p < 0.001 and 6.4 using IL-2 (p < 0.001, for autologous donor culture responses. For donor-versus-recipient culture responses, the increase was by a factor of 1.9 using IL-4 (p < 0.001 and 4.1 using IL-2 (p < 0.001. For donor-versus-unrelated culture responses, no significant increase was observed using IL-4, and a mean response inhibition of 20% was observed using IL-2 (p < 0.001. Neither of the cytokines produced a significant difference in the unrelated control versus recipient cell responses. CONCLUSION: IL-4 supplementation was the best for increasing the mixed lymphocyte culture sensitivity. However, IL-4 also increased autologous responses, albeit less

Differentiation of bone marrow cells with irradiated bone in vitro

International Nuclear Information System (INIS)

Toshiyuki Tominaga; Moritoshi Itoman; Izumi, T.; Wakita, R.; Uchino, M.

1999-01-01

Disease transmission or infection is an important issue in bone allograft, and irradiation is used for sterilization of graft bones. One of the advantages of bone allograft over biomaterials is that graft bones have osteoinductive factors such as growth factors. Irradiation is reported to decrease the osteoinductive activity in vivo. We investigated the osteoinductive activity of irradiated bone by alkaline phosphatase (ALP) activity in rat bone marrow cell culture. Bones (tibias and femurs of 12-week-old Wistar rats) were cleaned of adhering soft tissue, and the marrow was removed by washing. The bones were defatted, lyophilized, and cut into uniform 70 mg fragments. Then the Bone fragments were irradiated at either 10, 20, 25, 30, 40, or 50 kGy at JAERI. Bone marrow cells were isolated from tibias and femurs of 4-week-old Wistar rats. Cells were plated in tissue culture flask. When primary cultures reached confluence, cells were passaged (4 x 103 cell / cm2) to 6 wells plates. The culture medium consisted of minimum essential medium, 10% fetal bovine serum, ascorbic acid, and antibiotics. At confluence, a cell culture insert was set in the well, and an irradiated bone fragment was placed in it. Then, medium was supplemented with 10 mM ?-glycerophosphate and 1 x 10-8 M dexamethasone. Culture wells were stained by naphthol AS-MX phosphate, N,N-dimethyl formamide, Red violet LB salt on day 0, 7, 14. The density of ALP staining was analyzed by a personal computer. Without bones, ALP staining increased by 50% on day 7 and by 100% on day 14, compared with that on day 0. The other side, with bones irradiated at 30 kGy or lower, ALP staining increased by 150% on day 7, and by 180% on day 14, compared with that on day 0. In the groups of irradiated bones of 40 kGy or higher, the increase in ALP staining was less prominent compared with the groups of irradiated bones of 30 kGy or lower. In the groups of 0-30 kGy irradiation, ALP staining increased in the early period

Effects of bone morphogenetic protein-2 on bone cells in primary culture: immunohistochemical and electronmicroscopical studies

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Schmitz, I.; Prochnow, N.; Mueller, K.M. [Berufsgenossenschaftliche Kliniken Bergmannsheil, Bochum (Germany). Inst. fuer Pathologie; Wiemann, M.; Schirrmacher, K.; Bingmann, D. [Essen Univ. (Germany). Inst. fuer Physiologie; Sebald, W. [Wuerzburg Univ. (Germany). Inst. fuer Physiologische Chemie II

2001-02-01

Bone morphogenetic protein 2 (BMP-2), among other morphogenetic effects on non osseous tissues, promotes bone formation in vivo. Therefore, BMP-2 may accelerate the integration of osseous implants. Although the effects of BMPs on cell proliferation have been studied extensively in vivo or in cell lines, little is published about effects on bone cells in primary cultures, especially on cell differentiation. As such information is a prerequisite to understand and to control effects of BMPs on cells at the surface of implant materials, the present experiments aimed to describe effects of BMP-2 on primary cultures derived from calvarial fragments of neonatal rats. The cells were stimulated with 50 nM BMP-2 added to the nutrient medium for 3 or 6 days. Light- and electronmicroscopical studies showed that cells in the sprouting zones were larger and more often spindle shaped. Stimulated cells had more nucleoli than control cells and the endoplasmic reticulum was widened. They retained properties of typical bone cells: An immunhistochemical analysis showed that stimulated cells increased the activity of alkaline phosphatase, they secreted collagen type I and to a minor extent collagen type III. In BMP-2 treated cells the pattern of cells stained for actin, desmin and vimentin hardly changed whereas extracellular fibronectin appeared to be less cross-linked in BMP-2 treated cultures. The distribution and labeling strength of osteocalcin, a specific marker protein of bone cells did not change markedly. After exposure to BMP-2 cells tended to detach from the cover slips. Electron microscopy showed a reduced number of cell processes possibly facilitating the detachment and/or mobility. Stimulated cells contained an increased number of lamellar bodies which may reflect an increased synthesis and/or membrane turnover. Staining of non-osseous cells with anti-CD68-or anti-myeloid antibodies revealed that the small percentage of these cells regularly occurring in primary cultures

N-glycosylation profile of undifferentiated and adipogenically differentiated human bone marrow mesenchymal stem cells: towards a next generation of stem cell markers.

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Hamouda, Houda; Ullah, Mujib; Berger, Markus; Sittinger, Michael; Tauber, Rudolf; Ringe, Jochen; Blanchard, Véronique

2013-12-01

Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.

Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: the effect of silanol groups.

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Rodrigues, Márcia T; Leonor, Isabel B; Gröen, Nathalie; Viegas, Carlos A; Dias, Isabel R; Caridade, Sofia G; Mano, João F; Gomes, Manuela E; Reis, Rui L

2014-10-01

Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Leukemic blast cell colony formation in semisolid culture with erythropoietin: a case report of acute poorly differentiated erythroid leukemia.

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Tomonaga, M; Jinnai, I; Tagawa, M; Amenomori, T; Nishino, K; Yao, E; Nonaka, H; Kuriyama, K; Yoshida, Y; Matsuo, T

1987-02-01

The bone marrow of a patient with acute undifferentiated leukemia developed unique colonies after a 14-day culture in erythropoietin (EPO)-containing methylcellulose. The colonies consisted of 20 to 200 nonhemoglobinized large blast cells. Cytogenetic analysis of single colonies revealed hypotetraploid karyotypes with several marker chromosomes that were identical to those found in directly sampled bone marrow. The concurrently formed erythroid bursts showed only normal karyotypes. No leukemic colony formation was observed in other culture systems with either colony-stimulating activity (CSA) or phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). The leukemic colonies exhibited a complete EPO-dose dependency similar to that of the patient's normal BFU-E. Although cytochemical and immunologic marker studies of the bone marrow cells failed to clarify the cell lineage of the leukemic cells with extraordinarily large cell size, ultrastructural study revealed erythroid differentiation such as siderosome formation in the cytoplasm and ferritin particles in the rhophecytosis invaginations. These findings indicate that the patient had poorly differentiated erythroid leukemia and that some of the clonogenic cells might respond to EPO in vitro. Corresponding to this biological feature, the leukemic cells were markedly decreased in number in response to repeated RBC transfusions, and partial remission was obtained. These observations suggest that erythroid leukemia distinct from erythroleukemia (M6) with a myeloblastic component, can develop as a minor entity of human acute leukemia.

Hydroxyapatite/collagen bone-like nanocomposite.

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Kikuchi, Masanori

2013-01-01

Our group has succeeded to synthesize material with bone-like nanostructure and bone-like inorganic and organic composition via self-organization mechanism between them using simultaneous titration method under controlled pH and temperature. The hydroxyapatite/collagen (HAp/Col) bone-like nanocomposite completely incorporated into bone remodeling process to be substituted by new bone. Cells cultured on the HAp/Col revealed very interesting reactions. Osteoblast-like MG63 cells showed upregulation of alkaline phosphatase >3 times greater than MG63 cells cultured on tissue culture polystyrene (TCPS). MG63 cells 3-dimensionally cultured in a "HAp/Col sponge," a porous HAp/Col having sponge-like viscoelasticity, accumulated calcium phosphate nodules on extracellular matrices they secreted. Bone marrow cells co-cultured with osteoblasts on HAp/Col differentiated to osteoclasts without differentiation supplements. This phenomenon is not found in cells cultured on hydroxyapatite ceramics and TCPS, and rarely in cells cultured on dentin. These results suggest that HAp/Col is a good candidate for tissue engineering of bone as well as bone filler. In a clinical test as a bone filler, the HAp/Col sponge was significantly better than porous β-tricalcium phosphate. The HAp/Col sponge has been approved by the Japanese government and will be used as greatly needed bone filler in patients. In addition to the above, HAp/Col coating on titanium revealed higher osteo-conductivity than HAp-coated titanium and bare titanium and improved direct bonding between titanium and newly formed bone. The HAp/Col coating may be used for metal devices requiring osseointegration.

Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

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Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

2016-02-17

Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Organotypic culture of human bone marrow adipose tissue.

Science.gov (United States)

Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji

2010-04-01

The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

3D printed scaffolds of calcium silicate-doped β-TCP synergize with co-cultured endothelial and stromal cells to promote vascularization and bone formation.

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Deng, Yuan; Jiang, Chuan; Li, Cuidi; Li, Tao; Peng, Mingzheng; Wang, Jinwu; Dai, Kerong

2017-07-17

Synthetic bone scaffolds have potential application in repairing large bone defects, however, inefficient vascularization after implantation remains the major issue of graft failure. Herein, porous β-tricalcium phosphate (β-TCP) scaffolds with calcium silicate (CS) were 3D printed, and pre-seeded with co-cultured human umbilical cord vein endothelial cells (HUVECs) and human bone marrow stromal cells (hBMSCs) to construct tissue engineering scaffolds with accelerated vascularization and better bone formation. Results showed that in vitro β-TCP scaffolds doped with 5% CS (5%CS/β-TCP) were biocompatible, and stimulated angiogenesis and osteogenesis. The results also showed that 5%CS/β-TCP scaffolds not only stimulated co-cultured cells angiogenesis on Matrigel, but also stimulated co-cultured cells to form microcapillary-like structures on scaffolds, and promoted migration of BMSCs by stimulating co-cultured cells to secrete PDGF-BB and CXCL12 into the surrounding environment. Moreover, 5%CS/β-TCP scaffolds enhanced vascularization and osteoinduction in comparison with β-TCP, and synergized with co-cultured cells to further increase early vessel formation, which was accompanied by earlier and better ectopic bone formation when implanted subcutaneously in nude mice. Thus, our findings suggest that porous 5%CS/β-TCP scaffolds seeded with co-cultured cells provide new strategy for accelerating tissue engineering scaffolds vascularization and osteogenesis, and show potential as treatment for large bone defects.

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

International Nuclear Information System (INIS)

Yang Chiming; Frei, Hanspeter; Burt, Helen M.; Rossi, Fabio

2009-01-01

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.

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

International Nuclear Information System (INIS)

McCulloch, C.A.; Fair, C.A.; Tenenbaum, H.C.; Limeback, H.; Homareau, R.

1990-01-01

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

Bone marrow extract as a growth supplement for human iliac apophyseal chondrocyte culture

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Balasubramanian Balakumar

2016-01-01

Full Text Available Background & objectives: Human bone marrow is rich in various growth factors which may support the chondrocyte growth. This study was conducted to compare the culture characteristics of human growth plate chondrocyte in foetal bovine serum (FBS and human autologous bone marrow extract (BME in monolayer culture. Methods: Iliac crest apophyseal cartilage was harvested from four donors, aged between two and nine years, undergoing hip surgery. Chondrocytes were propagated under two culture conditions, with 10 per cent FBS and 10 per cent autologous BME harvested from the same donors. Cells were harvested at 7, 14 and 21 days to assess viability, morphology, cell count and immunocytochemistry. Results: With an initial seeding density of 2500 cells/cm 2 , the average yield in monolayer cultured with FBS was 3.35 × 10 5 , 5.9 × 10 5 , 14.1 × 10 5 and BME was 0.66 × 10 5 , 1.57 × 10 5 and 3.48 × 10 5 at 7, 14 and 21 days, respectively. Viability was 98.21 per cent with FBS and 97.45 per cent with BME at 21 days. In BME supplemented cultures, hyaline phenotype was maintained up to 21 days. The yield was higher in the FBS supplemented group; however, the phenotype could not be maintained by the FBS group as long as BME group. Interpretation & conclusions: Autologous BME was found to be a safer alternative to FBS for human studies. BME could maintain the hyaline phenotype for a longer time. Ways to enhance the cell yield needs to be explored in future studies.

The osteogenic response of undifferentiated human mesenchymal stem cells (hMSCs) to mechanical strain is inversely related to body mass index of the donor.

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Friedl, Gerald; Windhager, Reinhard; Schmidt, Helena; Aigner, Reingard

2009-08-01

While the importance of physical factors in the maintenance and regeneration of bone tissue has been recognized for many years and the mechano-sensitivity of bone cells is well established, there is increasing evidence that body fat constitutes an independent risk factor for complications in bone fracture healing and aseptic loosening of implants. Although mechanical causes have been widely suggested, we hypothesized that the osteogenic mechano-response of human mesenchymal stem cells (hMSCs) may be altered in obese patients. We determined the phenotypic and genotypic response of undifferentiated hMSCs of 10 donors to cyclic tensile strain (CTS) under controlled in vitro conditions and analyzed the potential relationship relevant to the donor's anthropomorphometric and biochemical parameters related to donor's fat and bone metabolism. The osteogenic marker genes were all statistically significantly upregulated by CTS, which was accompanied by a significant increase in cell-based ALP activity. Linear correlation analysis revealed that there was a significant correlation between phenotypic CTS response and the body mass index of the donor (r = -0.91, p < 0.001) and phenotypic CTS response was also significantly related to leptin levels (r = -0.68) and estradiol levels (r = 0.67) within the bone marrow microenvironment of the donor. Such an upstream imprinting process mediated by factors tightly related to the donor's fat metabolism, which hampers the mechanosensitivity of hMSCs in obese patients, may be of pathogenetic relevance for the complications associated with obesity that are seen in orthopedic surgery.

Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells

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Akporiaye, E T; Stewart, S; Stewart, C C

1984-01-01

Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. The authors report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc ..gamma.. receptors, and had esterase activity. 23 references, 1 figure, 1 table.

FEATURES OF CLINICAL COURSE OF GASTROESOPHAGEAL REFLUX DISEASE IN NEWLY RECRUITED WITH CONNECTIVE TISSUE UNDIFFERENTIATED DYSPLASIA SYNDROME

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E.I. Kashkina

2008-12-01

Full Text Available The presence of connective tissue undifferentiated dysplasia syndrome against a background of psychological stress at newly recruited can promote the risk of gastroesophageal reflux disease occurrence. To the utmost, correlation between the gastroesophageal reflux disease and such manifestations of connective tissue undifferentiated dysplasia syndrome as asthenic constitution, chest deformation, Gothic palate and hypermobility of joints was found

Directed Secretion by Bone Cells of a Factor that Attracts Breast Cancer Cells

National Research Council Canada - National Science Library

Gay, Carol

2001-01-01

The hFOB osteoblast cell line was cultured in both undifferentiated and differentiated states and tested for the capacity of the cell layers to occlude fluorescent-tagged dextrans of 4-, 20- and 40 kD molecular weight...

Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures.

Science.gov (United States)

Cerdan, Chantal; Hong, Seok Ho; Bhatia, Mickie

2007-10-01

The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein-4 (BMP-4) was able to generate up to 90% of CD45(+) hematopoietic cells with colony-forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies.

Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

Science.gov (United States)

Magnusson, Mattias; Sierra, Maria I; Sasidharan, Rajkumar; Prashad, Sacha L; Romero, Melissa; Saarikoski, Pamela; Van Handel, Ben; Huang, Andy; Li, Xinmin; Mikkola, Hanna K A

2013-01-01

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

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Mattias Magnusson

Full Text Available Lack of HLA-matched hematopoietic stem cells (HSC limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC stroma that protects human hematopoietic stem/progenitor cells (HSPC from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+ characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: In vitro cell culture studies

International Nuclear Information System (INIS)

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

2014-01-01

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. - Highlights: • Hydroxyapatite/poly(ε-caprolactone) scaffold with interconnected pores was prepared • Cytotoxicity test showed that the scaffold was not cytotoxic towards MC3T3-E1 cells • The scaffold supported the attachment, proliferation and differentiation of cells • A 3D cell colonization was confirmed using the fluorescence microscopy • The scaffold might be a promising candidate for bone tissue engineering

Cytokine and immunoglobulin production by PWM-stimulated peripheral and tumor-infiltrating lymphocytes of undifferentiated nasopharyngeal carcinoma (NPC) patients

International Nuclear Information System (INIS)

Fliss-Jaber, Lilia; Houissa-Kastally, Radhia; Bouzouita, Kamel; Khediri, Naceur; Khelifa, Ridha

2004-01-01

Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA). Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant. The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002), IL-10 (p = 0,020), IgM (p= 0,0003) and IgG (p < 0,0001) than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 (p = 0,022), IL-10 (p = 0,016) and IgM (p = 0,004) than those of controls. No significant differences for IL-6 and IgA were observed. Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC

Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application.

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Stefan Stich

Full Text Available For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation, 3.9% bipotent (without chondrogenic differentiation, and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

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Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

2018-01-06

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Pre-irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long-term bone marrow cultures

International Nuclear Information System (INIS)

Rooney, P.; Wright, E.G.

1993-01-01

Empty plastic tissue culture flasks were exposed to X-irradiation doses of 0.3-10.0 Gy, prior to the establishment of long-term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non-adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non-adherent cells produced. Histological examination of non-adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X-irradiation of glass tissue culture flasks had no deleterious effect. (author)

Selective inhibition of B lymphocytes in TBTC-treated human bone marrow long-term culture.

NARCIS (Netherlands)

Carfi', M.; Bowe, G.; Pieters, R.; Gribaldo, L.

2010-01-01

Tributyltin chloride (TBTC) is well known for its immunotoxic effect, in particular towards immature thymocytes. TBTC is also known to induce adipocyte differentiation in primary human bone marrow cultures, which is reflected in the decrease in a number of adipocyte-derived cytokines, chemokines and

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

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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.

In vitro cultures of Salvia officinalis L. as a source of antioxidant compounds

Directory of Open Access Journals (Sweden)

Izabela Grzegorczyk

2011-01-01

Full Text Available The concentrations of carnosic acid, carnosol and rosmarinic acid in different materials from differentiated (multiple shoot cultures and regenerated plants and undifferentiated (callus and cell suspension in vitro cultures of Salvia officinalis were determined by HPLC. The results suggested that diterpenoid (carnosic acid and carnosol production is closely related to shoot differentiation. The highest diterpenoid yield (11.4 mg g-1 for carnosic acid and 1.1 mg g-1 for carnosol was achieved in shoots of 10-week-old micropropagated plants. The levels were comparable to those found in shoots of naturally growing plants. Undifferentiated callus and cell suspension cultures produced only very low amounts of carnosol (ca. 0.05 mg g-1 of dry weight. In contrast, content of rosmarinic acid in callus and suspension cultures as well as shoots growing in vitro and in vivo was similar and ranged between 11.2 and 18.6 mg g-1 of dry weight.

Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson's disease.

Directory of Open Access Journals (Sweden)

Mariana eAcquarone

2015-04-01

Full Text Available Parkinson’s disease (PD is an incurable progressive neurodegenerative disorder. Clinical presentation of PD stems largely from the loss of dopaminergic neurons in the nigrostriatal dopaminergic pathway, motivating experimental strategies aimed at replacing dopaminergic innervation by cellular therapy. Transplantation of dopaminergic neurons derived from embryonic stem cells significantly improves motor functions in rodent and non-human primate models of PD. However, protocols to generate dopaminergic neurons from embryonic stem cells generally meet with low efficacy and high risk of teratoma development upon transplantation. To address these issues, we have pre-treated undifferentiated mouse embryonic stem cells (mESCs with the DNA alkylating agent mitomycin C (MMC before transplantation. MMC treatment of cultures prevented tumor formation in a 12-week follow-up after mESCs were injected in nude mice. In 6-OH-dopamine-lesioned mice, intrastriatal injection of MMC-treated mESCs markedly improved motor function without tumor formation for as long as 15 months. Furthermore, we show that halting mitotic activity of undifferentiated mESCs induces a four-fold increase in dopamine release following in vitro differentiation. Our findings indicate that treating mESCs with mitomycin C prior to intrastriatal transplant is an effective strategy that could be further investigated as a novel alternative for treatment of Parkinson's disease.

Fluctuating levels of reprogramming factor expression in cultured ...

African Journals Online (AJOL)

Although human undifferentiated keratinocytes (HUKs) can be reprogrammed to become induced pluripotent stem cells (iPSCs) with high efficiency and rapid kinetics by transducing reprogramming factors (RFs), the endogenous expression of reprogramming factors in cultured HUKs is not clear at different stages. In this ...

Postradiation recovery of human bone marrow, and morphological dynamics of undifferentiated cell pool

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Suvorova, L.A.; Vyalova, N.A.; Barabanova, A.V.; Gruzdev, G.P.

1981-09-01

The results of clinical follow-up of a group of subjects who had been exposed to uncontrolled radiation in accident situations were published in the Soviet literature: 1 patient was exposed to relative uniform gamma radiation, 8 to nonuniform gamma-neutron radiation in doses of the order of 2-5 Gy or more. Summarized are the hematological data referable to the same clinical observations and details on histological structural distinctions of bone marrow, using quantitative characteristics to describe the behavior of different hemopoietic stem cells.

Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro

International Nuclear Information System (INIS)

Mori, K.J.; Izumi, Hiroko; Seto, Akira

1981-01-01

A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent 'stromal' cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied. (author)

Etiologies of Acute Undifferentiated Fever and Clinical Prediction of Scrub Typhus in a Non-Tropical Endemic Area

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Jung, Ho-Chul; Chon, Sung-Bin; Oh, Won Sup; Lee, Dong-Hyun; Lee, Ho-Jin

2015-01-01

Scrub typhus usually presents as acute undifferentiated fever. This cross-sectional study included adult patients presenting with acute undifferentiated fever defined as any febrile illness for ≤ 14 days without evidence of localized infection. Scrub typhus cases were defined by an antibody titer of a ≥ fourfold increase in paired sera, a ≥ 1:160 in a single serum using indirect immunofluorescence assay, or a positive result of the immunochromatographic test. Multiple regression analysis identified predictors associated with scrub typhus to develop a prediction rule. Of 250 cases with known etiology of acute undifferentiated fever, influenza (28.0%), hepatitis A (25.2%), and scrub typhus (16.4%) were major causes. A prediction rule for identifying suspected cases of scrub typhus consisted of age ≥ 65 years (two points), recent fieldwork/outdoor activities (one point), onset of illness during an outbreak period (two points), myalgia (one point), and eschar (two points). The c statistic was 0.977 (95% confidence interval = 0.960–0.994). At a cutoff value ≥ 4, the sensitivity and specificity were 92.7% (79.0–98.1%) and 90.9% (86.0–94.3%), respectively. Scrub typhus, the third leading cause of acute undifferentiated fever in our region, can be identified early using the prediction rule. PMID:25448236

Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.

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Poudel, Sher Bahadur; Bhattarai, Govinda; Kook, Sung-Ho; Shin, Yun-Ji; Kwon, Tae-Ho; Lee, Seung-Youp; Lee, Jeong-Chae

2017-10-01

Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

A new and efficient culture method for porcine bone marrow-derived M1- and M2-polarized macrophages.

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Gao, Jiye; Scheenstra, Maaike R; van Dijk, Albert; Veldhuizen, Edwin J A; Haagsman, Henk P

2018-06-01

Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

NUTM1 Gene Fusions Characterize a Subset of Undifferentiated Soft Tissue and Visceral Tumors.

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Dickson, Brendan C; Sung, Yun-Shao; Rosenblum, Marc K; Reuter, Victor E; Harb, Mohammed; Wunder, Jay S; Swanson, David; Antonescu, Cristina R

2018-05-01

NUT midline carcinoma is an aggressive tumor that occurs mainly in the head and neck and, less frequently, the mediastinum and lung. Following identification of an index case of a NUTM1 fusion positive undifferentiated soft tissue tumor, we interrogated additional cases of primary undifferentiated soft tissue and visceral tumors for NUTM1 abnormalities. Targeted next-generation sequencing was performed on RNA extracted from formalin-fixed paraffin-embedded tissue, and results validated by fluorescence in situ hybridization using custom bacterial artificial chromosome probes. Six patients were identified: mean age of 42 years (range, 3 to 71 y); equal sex distribution; and, tumors involved the extremity soft tissues (N=2), kidney (N=2), stomach, and brain. On systemic work-up at presentation all patients lacked a distant primary tumor. Morphologically, the tumors were heterogenous, with undifferentiated round-epithelioid-rhabdoid cells arranged in solid sheets, nests, and cords. Mitotic activity was generally brisk. Four cases expressed pancytokeratin, but in only 2 cases was this diffuse. Next-generation sequencing demonstrated the following fusions: BRD4-NUTM1 (3 cases), BRD3-NUTM1, MXD1-NUTM1, and BCORL1-NUTM1. Independent testing by fluorescence in situ hybridization confirmed the presence of NUTM1 and partner gene rearrangement. This study establishes that NUT-associated tumors transgress the midline and account for a subset of primitive neoplasms occurring in soft tissue and viscera. Tumors harboring NUTM1 gene fusions are presumably underrecognized, and the extent to which they account for undifferentiated mesenchymal, neuroendocrine, and/or epithelial neoplasms is unclear. Moreover, the relationship, if any, between NUT-associated tumors in soft tissue and/or viscera, and conventional NUT carcinoma, remains to be elucidated.

Is undifferentiated spondyloarthritis a discrete entity? A debate.

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Deodhar, Atul; Miossec, Pierre; Baraliakos, Xenofon

2018-01-01

The concept of undifferentiated spondyloarthritis has been introduced recently to describe a clinical setting where the classical features of spondyloarthritis (SpA) are not fully present. Whether this is a discrete entity was the basis of a debate during the 4th International Congress on Controversies in Rheumatology & Autoimmunity held in Bologna, Italy 9-11 March 2017. The pro and con aspects of the debate are presented. The implications of the debate are important ranging from diagnostic aspects to consequences for the society and the payers. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell Culturing of Cytoskeleton

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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.

Survival of human mesenchymal stromal cells from bone marrow and adipose tissue after xenogenic transplantation in immunocompetent mice

DEFF Research Database (Denmark)

Niemeyer, P; Vohrer, J; Schmal, H

2008-01-01

of the current paper was to evaluate the survival of undifferentiated and osteogenically induced human MSC from different origins after transplantation in immunocompetent mice. METHODS: Human MSC were isolated from bone marrow (BMSC) and adipose tissue (ASC). After cultivation on mineralized collagen, MSC were......INTRODUCTION: Mesenchymal stromal cells (MSC) represent an attractive cell population for tissue engineering purposes. As MSC are described as immunoprivileged, non-autologous applications seem possible. A basic requirement is the survival of MSC after transplantation in the host. The purpose...... transplanted subcutaneously into immunocompetent mice (n=12). Undifferentiated MSC (group A) were compared with osteogenic-induced MSC (group B). Human-specific in situ hybridization and anti-vimentin staining was used to follow MSC after transplantation. Quantitative evaluation of lymphocytes and macrophages...

Co-cultured hBMSCs and HUVECs on human bio-derived bone scaffolds provide support for the long-term ex vivo culture of HSC/HPCs.

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Huang, Xiaobing; Li, Chenglong; Zhu, Biao; Wang, Hailian; Luo, Xiangwei; Wei, Lingling

2016-05-01

In order to closely mimic a multi-cell state in hematopoietic stem/progenitor cells (HSC/HPCs) vascular niche, we co-cultured human bone marrow mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) without any cytokines as feeder cells and applied bio-derived bone from human femoral metaphyseal portion as scaffold to develop a new HSC/HPCs three-dimensional culture system (named 3D-Mix cultures). Scanning electron and fluorescent microscopy showed excellent biocompatibility of bio-derived bone to hBMSCs and HUVECs in vitro. Flow cytometry analysis and quantitative real-time polymerase chain reaction (qPCR) assay of p21 expression demonstrated that 3D-Mix could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than 3D-hMSC and 3D-HUVEC. Long-term culture initiating cell (LTC-IC) confirmed that 3D-Mix had the most powerful activity of maintaining multipotent differentiation of primitive cell subpopulation in HSCs. The nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cell (SRC) assay demonstrated that 3D-Mix promoted the expansion of long-term primitive transplantable HSCs. qPCR of alkaline phosphatase (ALP) and osteocalcin (OC) demonstrated that HUVECs enhanced the early osteogenic differentiation of BMSCs. Western blot and qPCR revealed that HUVECs activated Wnt/β-catenin signaling in hBMSCs inducing Notch signal activation in HSCs. Our study indicated that interaction between hMSCs and HUVECs may have a critical role in to influent on HSCs/HPCs fate in vitro. These results demonstrated that the 3D-Mix have the ability to support the maintenance and proliferation of HSCs/HPCs in vitro. © 2016 Wiley Periodicals, Inc.

Osteogenesis and Morphology of the Peri-Implant Bone Facing Dental Implants

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Marco Franchi

2004-01-01

Full Text Available This study investigated the influence of different implant surfaces on peri-implant osteogenesis and implant face morphology of peri-implant tissues during the early (2 weeks and complete healing period (3 months. Thirty endosseous titanium implants (conic screws with differently treated surfaces (smooth titanium = SS, titanium plasma sprayed = TPS, sand-blasted zirconium oxide = Zr-SLA were implanted in femur and tibiae diaphyses of two mongrel sheep. Histological sections of the implants and surrounding tissues obtained by sawing and grinding techniques were observed under light microscopy (LM. The peri-implant tissues of other samples were mechanically detached from the corresponding implants to be processed for SEM observation. Two weeks after implantation, we observed osteogenesis (new bone trabeculae around all implant surfaces only where a gap was present at the host bone-metal interface. No evident bone deposition was detectable where threads of the screws were in direct contact with the compact host bone. Distance osteogenesis predominated in SS implants, while around rough surfaces (TPS and Zr-SLA, both distance and contact osteogenesis were present. At SEM analysis 2 weeks after implantation, the implant face of SS peri-implant tissue showed few, thin, newly formed, bone trabeculae immersed in large, loose, marrow tissue with blood vessels. Around the TPS screws, the implant face of the peri-implant tissue was rather irregular because of the rougher metal surface. Zr-SLA screws showed more numerous, newly formed bone trabeculae crossing marrow spaces and also needle-like crystals in bone nodules indicating an active mineralising process. After 3 months, all the screws appeared osseointegrated, being almost completely covered by a compact, mature, newly formed bone. However, some marrow spaces rich in blood vessels and undifferentiated cells were in contact with the metal surface. By SEM analysis, the implant face of the peri

Identification of Rorβ targets in cultured osteoblasts and in human bone

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

2013-11-01

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

Viral respiratory tract infections among patients with acute undifferentiated fever in Vietnam

NARCIS (Netherlands)

Phuong, Hoang Lan; Nga, Tran T. T.; van Doornum, Gerard J.; Groen, Jan; Binh, Tran Q.; Giao, Phan T.; Hung, Le Q.; Nams, Nguyen V.; Kager, P. A.; de Vries, Peter J.

2010-01-01

To investigate the proportion of viral respiratory tract infections among acute undifferentiated fevers (AUFs) at primary health facilities in southern Vietnam during 2001-2005, patients with AUF not caused by malaria were enrolled at twelve primary health facilities and a clinic for malaria control

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

Science.gov (United States)

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Progenitor cells of erythroblasts: an in vitro investigation of erythropoietin-responsive cells of guinea pig bone marrow

International Nuclear Information System (INIS)

Rosse, C.; Beaufait, D.W.

1978-01-01

The experiments were designed to therst whether erythroblast progenitor cell function could be demonstrated in a morphological cell type designated as transitional cells. Two cell fractions were obtained from the bone marrow of normal and polycythemic guinea pigs. One fraction (F1) was enriched in transitional cells and contained few other cell types which could be considered as candidates for erythropoietin responsive cells (ERC). The other fraction (F2) contained undifferentiated blast cells as well as transitional cells. The effect of human urinary erythropoiesis stimulating factors (ESF) on heme synthesis was compared in these two fractions by measuring 59 Fe incorporation into heme. ESF was more effective in stimulating heme synthesis in guinea pig bone marrow cells than homologous sera obtained from anemic or hypoxic animals. The majority of ERC sedimented in F2, but the stimulation index was comparable in the two fractions. It was confirmed by radioautography that the ESF response in F1 was due to the generation of proerythroblasts and basophilic erythroblasts that incorporated 55 Fe. The generation of these cells in F1 was dependent on the addition of ESF to the cultures, whereas 55 Fe-labeled erythroblasts were recovered from cultures of F2 not supplemented with ESF. ESF induced a proportion of transitional cells to incorporate 55 Fe in both F1 and F2. Transitional cells were the only cell type in which heme synthesis was dependent on ESF. Radioautography with 55 Fe identified a proportion of these cells as ERC in both F1 and F2 fractions of bone marrow obtained from normal and polycythemic guinea pigs. The present studies show that some transitional cells function as progenitors of erythroblasts because they respond to ESF by initiation of heme synthesis and by transformation into the earliest recognizable erythroid cells

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

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G Bouet

2015-05-01

Full Text Available An engineered three dimensional (3D in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a “proof-of-concept” for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

Characterization of acute undifferentiated leukemia by combined analysis of plasma membrane-associated gamma-glutamyltranspeptidase and soluble terminal transferase.

Science.gov (United States)

Heumann, D; Losa, G; Barras, C; Morell, A; von Fliedner, V

1985-08-01

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma-GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma-GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT-blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma-GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.

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

Science.gov (United States)

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

2010-06-01

Bone marrow-derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow-derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony-forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow-derived stem cells. (c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Prevalence of undifferentiated fever in adults of Rawalpindi having primary dengue fever

International Nuclear Information System (INIS)

Zafar, H.; Hayyat, A.; Akhtar, N.

2013-01-01

The objectives of the study were to highlight early subclinical presentation of dengue viral infection (DVI) as an undifferentiated febrile illness. The descriptive cross-sectional study was carried out at Microbiology Department, Rawalpindi Medical College from March to September 2009. Stratified random sampling was used to select subjects from various urban and rural areas of Rawalpindi, and Serum IgG anti-dengue antibodies were detected by using 3rd generation enzyme-linked immunosorbent assay (ELISA). Out of the total 240 subjects, 69 (28.75%) were found to be positive for anti-dengue IgG antibodies. Of the positive cases, 41 (59.4%) - comprising 31 (44.9%) urban residents - and 10 (14.4%) rural residents presented with a previous history of undifferentiated fever (p<0.05). It was concluded that primary DVI can present as subclinical form in healthy population residing in rural and urban areas of Rawalpindi, which is an alarming situation indicating the spread of disease in the study area. (author)

Effects of bacterial lipopolysaccharide and X-irradiation on the production of colony-stimulating factor and the maintenance of granulopoiesis in bone marrow culture

International Nuclear Information System (INIS)

Izumi, H.; Miyanomae, T.; Tsurusawa, M.; Fujita, J.; Mori, K.

1984-01-01

Effects of bacterial lipopolysaccharide (LPS) and X-irradiation on CSF production and granulopoiesis in long-term bone marrow cultures were studied. Levels of colony-stimulating factor (CSF) increased soon after the refeeding of the culture, but the activity was undetectable at day 7. Addition of LPS induced a significant increase in CSF levels in the culture, followed by an elevated granulopoiesis. The increase in CSF levels was suppressed when culture medium that had been harvested at refeeding on day 7 was added. Although irradiation did not increase CSF production, granulopoiesis was markedly stimulated shortly after irradiation. Thus granulopoiesis in long-term bone marrow culture may also be regulated by humoral factors such as CSF, and the culture system may represent the in vivo response to haemopoietic stimuli. (author)

Biomaterials and bone mechanotransduction

Science.gov (United States)

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

2001-01-01

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

Undifferentiated Embryonic Cell Transcription Factor 1 Regulates ESC Chromatin Organization and Gene Expression

NARCIS (Netherlands)

Kooistra, Susanne M.; van den Boom, Vincent; Thummer, Rajkumar P.; Johannes, Frank; Wardenaar, Rene; Tesson, Bruno M.; Veenhoff, Liesbeth M.; Fusetti, Fabrizia; O'Neill, Laura P.; Turner, Bryan M.; de Haan, Gerald; Eggen, Bart J. L.; O’Neill, Laura P.

2010-01-01

Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES

Diffusion chamber culture of mouse bone marrow cells, (1)

International Nuclear Information System (INIS)

Sigeta, Chiharu; Tanaka, Kimio; Kawakami, Masahito; Takahashi, Hiroshi; Ohkita, Takeshi

1980-01-01

Mouse bone marrow cells were cultured in diffusion chambers (DC) implanted in the peritoneal cavity of host mice. Host mice were subjected to (1) irradiation ( 60 Co 800 rad) and/or (2) phenylhydrazine induced anemia and then receiving irradiation ( 60 Co 600 rad). After culture periods of 3-7 days, the total number of cells in DC was increased. A marked increase in DC is due to the proliferation of granulocyte series. When host mice were subjected to anemia and irradiation, the start of cell proliferation in DC was delay about two days. On the whole, anemia and irradiation host reduced a little cell growth in DC. The number of immature granulocytes grown in DC in irradiated hosts or anemia and irradiated hosts increased and reached a plateu at day 5. During the plateu period, the proportions between immature and mature granulocytes in DC were kept constantly. The number of macrophages showed a two-phase increasing. Erythroid cells and lymphocytes rapidly disappeared from the chambers during 3 days. The number of erythroid cells was not significantly influenced even in anemia and irradiation hosts. (author)

Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation.

Science.gov (United States)

Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter

2011-07-14

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.

Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation

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Meyer Ulrich

2011-07-01

Full Text Available Abstract Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG. After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin.

Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

Science.gov (United States)

Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

2014-04-01

The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

Conditionally replicating adenovirus prevents pluripotent stem cell–derived teratoma by specifically eliminating undifferentiated cells

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Kaoru Mitsui

Full Text Available Incomplete abolition of tumorigenicity creates potential safety concerns in clinical trials of regenerative medicine based on human pluripotent stem cells (hPSCs. Here, we demonstrate that conditionally replicating adenoviruses that specifically target cancers using multiple factors (m-CRAs, originally developed as anticancer drugs, may also be useful as novel antitumorigenic agents in hPSC-based therapy. The survivin promoter was more active in undifferentiated hPSCs than the telomerase reverse transcriptase (TERT promoter, whereas both promoters were minimally active in differentiated normal cells. Accordingly, survivin-responsive m-CRA (Surv.m-CRA killed undifferentiated hPSCs more efficiently than TERT-responsive m-CRAs (Tert.m-CRA; both m-CRAs exhibited efficient viral replication and cytotoxicity in undifferentiated hPSCs, but not in cocultured differentiated normal cells. Pre-infection of hPSCs with Surv.m-CRA or Tert.m-CRA abolished in vivo teratoma formation in a dose-dependent manner following hPSC implantation into mice. Thus, m-CRAs, and in particular Surv.m-CRAs, represent novel antitumorigenic agents that could facilitate safe clinical applications of hPSC-based regenerative medicine.

Cell based bone tissue engineering in jaw defects

NARCIS (Netherlands)

Meijer, Gert J.; de Bruijn, Joost Dick; Koole, Ron; van Blitterswijk, Clemens

2008-01-01

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix.

Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

Science.gov (United States)

Stern, Amber Rath; Stern, Matthew M.; Van Dyke, Mark E.; Jähn, Katharina; Prideaux, Matthew; Bonewald, Lynda F.

2013-01-01

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38.[Q1] The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease. PMID:22668415

Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

NARCIS (Netherlands)

Lokhorst, H. M.; Lamme, T.; de Smet, M.; Klein, S.; de Weger, R. A.; van Oers, R.; Bloem, A. C.

1994-01-01

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion

Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

OpenAIRE

Hayam Abdel Meguid El Aggan; Mona Abdel Kader Salem; Nahla Mohamed Gamal Farahat; Ahmad Fathy El-Koraie; Ghaly Abd Al-Rahim Mohammed Kotb

2013-01-01

Introduction: Chronic allograft nephropathy (CAN) is a poorly understood clinico-pathological entity associated with chronic allograft loss due to immunologic and non-immunologic causes. It remains the leading cause of late allograft loss. Bone marrow derived stem cells are undifferentiated cells typically characterized by their capacity for self renewal, ability to give rise to multiple differentiated cellular population, including hematopoietic (HSCs) and mesenchymal stem cells (MSCs). Char...

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

International Nuclear Information System (INIS)

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

2010-01-01

Research highlights: → BM-EPCs and MSCs establish complex, self-organizing structures in co-culture. → Co-culture decreases proliferation by cellular self-regulatory mechanisms. → Co-cultured cells present an activated proangiogenic phenotype. → 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.

BCNT studies for application to the undifferentiated thyroid carcinoma

International Nuclear Information System (INIS)

Dagrosa, Maria A.; Viaggi, Mabel E.; Cabrini, Romulo L.; Juvenal, Guillermo J.; Pisarev, Mario A.; Garavaglia, Ricardo N.; Farias, Silvia S.; Belli, Carolina; Larripa, Irene; Gangitano, David

2000-01-01

Undifferentiated thyroid carcinoma (UTC) lacks an effective treatment. Boron neutron capture therapy (BNCT) is based on the selective uptake of 10 B-boronated compounds by some tumours, followed by irradiation with an appropriate neutron beam. The radioactive boron originated ( 11 B) decays releasing 7 Li, gamma rays and alpha particles, and these latter will destroy the tumour. In order to explore the possibility of applying BNCT to UTC we have studied the biodistribution of BPA. Animal Model: To develop an animal model of undifferentiated thyroid carcinoma (UTC), which may be useful to study of BNCT. The UTC human cell line ARO was implanted into the back of the nude mice. We performed successive passages in mouse after tumor culturing in order to obtain an animal model similar to the human tumor. We studied the kinetics and the tumoral histology, the capability to induce metastasis, the biokinetics of in vitro growth, as well as cytogenetic and molecular aspects. Histological specimens of tumor showed extensive viability with high mitotic activity. At 117 days, the tumors reached a size of 1700 mm 3 and showed a central necrotic portion with a thin layer of viable cells presence of micro metastasis could be observed in the lung. The kinetics of growth both in vivo and in vitro showed that when the number of passages in mouse increases the growth rate decreases. The cytogenetic and molecular studies did not show differences between the original line and the sublines that could explain this phenotypic change. Moreover, the cytogenetic studies proved that the ARO cell line and its sublines showed a complex clonal karyotype including structural alterations with deletions and translocations involving chromosomes 5, 7, 8, 9p, 11p, 17q 19p, and 20q that were consistent with earlier reported data in UTC. In vivo BNCT studies: ARO cells were transplanted into the scapular region of NIH nude mice, and after 2 weeks BPA (350 or 600 mg/kg bw) was injected via i.p. The

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Spermatogonial multiplication in the Chinese hamster. II. Cell cycle properties of undifferentiated spermatogonia

NARCIS (Netherlands)

Lok, D.; Jansen, M. T.; de rooij, D. G.

1983-01-01

The cell cycle properties of undifferentiated spermatogonia in the Chinese hamster were analysed by the fraction of labelled mitoses technique (FLM) in whole mounted seminiferous tubules. The minimum cell cycle time (Tc) was found to be c. 90 hr for the As and 87 hr for the Apr and Aal

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

International Nuclear Information System (INIS)

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

2010-01-01

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 γ 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-β1 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.

Superficial EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion: a new variant of Ewing-like tumors with locoregional lymph node metastasis.

Science.gov (United States)

Machado, Isidro; Cruz, Julia; Lavernia, Javier; Rubio, Luis; Campos, Jorge; Barrios, María; Grison, Camille; Chene, Virginie; Pierron, Gaelle; Delattre, Olivier; Llombart-Bosch, Antonio

2013-12-01

The present study describes a new case of EWSR1-negative undifferentiated sarcoma with CIC/DUX4 gene fusion. This case is similar to tumors described as primitive undifferentiated round cell sarcomas that occur mainly in the trunk and display an aggressive behavior. To our knowledge, this is the first report of such a tumor presenting locoregional lymph node metastasis. In view of previous studies that prove the existence of a particular variant of undifferentiated sarcoma with Ewing-like morphology and CIC/DUX-4 gene fusion, a search for this gene fusion in all undifferentiated round cell sarcomas should be considered if a conclusive diagnosis cannot be reached following other conventional studies. Although additional cases with more extensive follow-up studies are needed, we believe that EWSR1-negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion should be added to the list of new sarcoma variants with the possibility of lymph node metastasis.

Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

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Kawinthra Khwanraj

2015-01-01

Full Text Available The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson’s disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH in undifferentiated and retinoic acid- (RA- induced differentiated cells. The western blot results showed a gradual decrease in TH in undifferentiated cells and a gradual increase in TH in differentiated cells from days 4 to 10 after cell plating. Immunostaining revealed a gradual increase in TH along with neuritic outgrowth in differentiated cells on days 4 and 7 of RA treatment. For the study on cell susceptibility to MPP+ and the expression of apoptosis-related genes, MTT assay showed a decrease in cell viability to approximately 50% requiring 500 and 1000 μM of MPP+ for undifferentiated and RA-differentiated cells, respectively. Using real-time RT-PCR, treatment with 500 μM MPP+ led to significant increases in the Bax/Bcl-2 ratio, p53, and caspase-3 in undifferentiated cells but was without significance in differentiated cells. In conclusion, differentiated cells may be more suitable, and the shorter duration of RA differentiation may make the SH-SY5Y cell model more accessible.

Co-cultures and cell sheet engineering as relevant tools to improve the outcome of bone tissue engineering strategies

OpenAIRE

Pirraco, Rogério

2011-01-01

Taking into consideration the complex biology of bone tissue it is quite clear that the understanding of the cellular interactions that regulate the homeostasis and regeneration of this remarkable tissue is essential for a successful Tissue Engineering strategy. The in vitro study of these cellular interactions relies on co-culture systems, a tremendously useful methodology where two or more cell types are cultured at the same time. Such strategy increases the complexity of typ...

Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture

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L Tirkkonen

2013-01-01

Full Text Available Human adipose stem cells (hASCs have been recently used to treat bone defects in clinical practice. Yet there is a need for more optimal scaffolds and cost-effective approaches to induce osteogenic differentiation of hASCs. Therefore, we compared the efficiency of bone morphogenetic proteins (BMP-2 and BMP-7, vascular endothelial growth factor (VEGF, and osteogenic medium (OM for the osteo-induction of hASCs in 3D culture. In addition, growth factors were tested in combination with OM. Commercially available bioactive glass scaffolds (BioRestore and biphasic calcium phosphate granules (BoneCeramic were evaluated as prospective carriers for hASCs. Both biomaterials supported hASC-viability, but BioRestore resulted in higher cell number than BoneCeramic, whereas BoneCeramic supported more significant collagen production. The most efficient osteo-induction was achieved with plain OM, promoting higher alkaline phosphatase activity and collagen production than growth factors. In fact, treatment with BMP-2 or VEGF did not increase osteogenic differentiation or cell number significantly more than maintenance medium with either biomaterial. Moreover, BMP-7 treatment consistently inhibited proliferation and osteogenic differentiation of hASCs. Interestingly, there was no benefit from growth factors added to OM. This is the first study to demonstrate that OM enhances hASC-differentiation towards bone-forming cells significantly more than growth factors in 3D culture.

Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

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Sébastien Flajollet

Full Text Available In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.

Construction of human induced pluripotent stem cell-derived oriented bone matrix microstructure by using in vitro engineered anisotropic culture model.

Science.gov (United States)

Ozasa, Ryosuke; Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Yun, Hui-Suk; Nakano, Takayoshi

2018-02-01

Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

FMR1 epigenetic silencing commonly occurs in undifferentiated fragile X-affected embryonic stem cells.

Science.gov (United States)

Avitzour, Michal; Mor-Shaked, Hagar; Yanovsky-Dagan, Shira; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Levy-Lahad, Ephrat; Epsztejn-Litman, Silvina; Eiges, Rachel

2014-11-11

Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5'-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.

FMR1 Epigenetic Silencing Commonly Occurs in Undifferentiated Fragile X-Affected Embryonic Stem Cells

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Michal Avitzour

2014-11-01

Full Text Available Fragile X syndrome (FXS is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5′-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%. In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.

Acute undifferentiated fever in Binh Thuan province, Vietnam: imprecise clinical diagnosis and irrational pharmaco-therapy

NARCIS (Netherlands)

Phuong, Hoang L.; de Vries, Peter J.; Nagelkerke, Nico; Giao, Phan T.; Hung, Le Q.; Binh, Tran Q.; Nga, Tran T. Thanh; Nam, Nguyen V.; Kager, Piet A.

2006-01-01

OBJECTIVES: To describe the characteristics of patients consulting commune primary healthcare posts for acute undifferentiated fever not being malaria (AUF), and to explore the diagnostic and therapeutic responses of the healthcare workers. METHODS: All patients presenting with AUF at 12 commune

Clinical and biochemical manifestations of undifferentiated forms of connective tissue dysplasia in pregnant women with varicose veins of small pelvis

Directory of Open Access Journals (Sweden)

N.M. Shibelgut

2010-03-01

Full Text Available Research objective is to define the pathogenesis of varicous veins of small pelvis in women. at Ultrasonic investigation of venous system of small pelvis has been carried out in 290 pregnant women. It revealed 190 patients with varicose veins of small pelvis (VVSP. By means of V.M. Jakovleva's technique phenotypic menifestation of connective tissue dysplasia was determined in all pregnant women. Biochemical manifestations of connective tissue dysplasia were identified by sialic acid level in blood serum, daily excretion of glycosaminoglycans and oxyproline. High frequency of clinical and biochemical manifestations of undifferentiated forms of connective tissue dysplasia was revealed in pregnant women with VVSP. Patients with VVSP developed tooth and jaw, facial and locomotor damages. Patients with VVSP characterized by visceral undifferentiated forms of connective tissue dysplasia demonstrated by refraction involvement, ventral hernias, flat feet, varicous veins of lower extremities, hypermobile syndrome, mitral valve prolapse of different degree. Biochemical manifestations of undifferentiated forms of connective tissue dysplasia in pregnant women with VVSP were insignificant

Effect of tryptophan hydroxylase gene polymorphism on aggression in major depressive disorder and undifferentiated somatoform disorder.

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Koh, Kyung Bong; Kim, Chan Hyung; Choi, Eun Hee; Lee, Young-joon; Seo, Won Youl

2012-05-01

Aggression and anger have been linked with depression, and anger suppression has been linked with somatic symptoms of somatoform disorders. However, the relationship between aggression or anger and genes in patients with depression and somatoform disorders has not been clearly elucidated. The objective of this study was to examine the effect of serotonin-related gene polymorphism on aggression in depressive disorders and somatoform disorders. A serotonin-related polymorphic marker was assessed by using single nucleotide polymorphism (SNP) genotyping. 106 outpatients with major depressive disorder (MDD), 102 outpatients with undifferentiated somatoform disorder, and 133 healthy subjects were enrolled between October 2005 and May 2008. Diagnoses were made according to the Korean version of the Structured Clinical Interview Schedule for DSM-IV. The allele and genotype frequencies of tryptophan hydroxylase-1 (TPH1) A218C were compared between groups. The Hamilton Depression Rating Scale and the Aggression Questionnaire were used for psychological assessment. Each of the 2 disorder groups scored significantly higher on all the Aggression Questionnaire subscales and on the total Aggression Questionnaire score than the healthy subjects (P sex and age. However, no significant differences were found in TPH1 C allele and CC homozygote frequencies between the undifferentiated somatoform disorder patients and the healthy subjects. TPH1 CC homozygote in the MDD group scored significantly higher in terms of verbal aggression (P = .03) and total Aggression Questionnaire score (P = .04) than A-carrier genotypes, regardless of sex and age. However, no significant differences were found in the scores of all the Aggression Questionnaire subscales and the total Aggression Questionnaire score between TPH1 CC homozygote and A-carrier genotypes in the undifferentiated somatoform disorder group and the control group, respectively. Aggression in MDD patients is more susceptible to an

Postirradiation changes in the amount and contact properties of bone marrow neutrophils in a short-term culture

International Nuclear Information System (INIS)

Chukhlovin, A.B.; Nikolaevskaya, L.V.

1988-01-01

A study was made of the adherence to plastic Petri dishes and viability ( as non-pyknotic cell counts) of rat bone marrow cells cultured for 5 to 22 h in 199 media containing 15% fresh isologous serum. An overall decrease in the number of viable myelokaryocytes including mature neutrophils was observed in the cultures irradiated with doses of 3 to 12 Gy. In addition gamma irradiation increased substantially the adherence of neutrophyls and to a lesser extent of other myelokaryocytes. A possibility of early radiation-induced disturbances in granulocytic maturation is discussed

TBTC induces adipocyte differentiation in human bone marrow long term culture

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Carfi, M.; Croera, C.; Ferrario, D.; Campi, V.; Bowe, G.; Pieters, R.; Gribaldo, L.

2008-01-01

Organotins are widely used in agriculture and the chemical industry, causing persistent and widespread pollution. Organotins may affect the brain, liver and immune system and eventually human health. Recently, it has been shown that tri-butyltin (TBT) interacts with nuclear receptors PPARγ (peroxisome proliferator-activated receptor γ) and RXR (retinoid x receptor) leading to adipocyte differentiation in the 3T3 cell line. Since adipocytes are known to influence haematopoiesis, for instance through the expression of cytokines and adhesion molecules, it was considered of interest to further study the adipocyte-stimulating effect of TBTC in human bone marrow cultures. Nile Red spectrofluorimetric analysis showed a significant increase of adipocytes in TBTC-treated cultures after 14 days of long term culture. Real-time PCR and Western blot analysis confirmed the high expression of the specific adipocyte differentiation marker aP2 (adipocyte-specific fatty acid binding protein). PPARγ, but not RXR, mRNA was increased after 24 h and 48 h exposure. TBTC also induced a decrease in a number of chemokines, interleukins, and growth factors. Also the expression of leptin, a hormone involved in haematopoiesis, was down regulated by TBTC treatment. It therefore appears that TBTC induced adipocyte differentiation, whilst reducing a number of haematopoietic factors. This study indicates that TBTC may interfere in the haematopoietic process through an alteration of the stromal layer and cytokine homeostasis

Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

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Saburo Hidaka

2006-01-01

Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

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

International Nuclear Information System (INIS)

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

1985-01-01

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

Myeloblastic and lymphoblastic markers in acute undifferentiated leukemia and chronic myelogenous leukemia in blast crisis.

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Shumak, K H; Baker, M A; Taub, R N; Coleman, M S

1980-11-01

Blast cells were obtained from 17 patients with acute undifferentiated leukemia and 13 patients with chronic myelogenous leukemia in blast crisis. The blasts were tested with anti-i serum in cytotoxicity tests and with antisera to myeloblastic leukemia-associated antigens in immunofluorescence tests. The terminal deoxynucleotidyl transferase (TDT) content of the blasts was also measured. Lymphoblasts react strongly with anti-i, do not react with anti-myeloblast serum, and have high levels of TDT; myeloblasts react weakly with anti-i, do not react with anti-myeloblast serum, and have very low levels of TDT. Of the 17 patients with acute undifferentiated leukemia, there were six with blasts which reacted like lymphoblasts, six with blasts which reacted like myeloblasts, and five with blasts bearing different combinations of these lymphoblastic and myeloblastic markers. Eight of the 11 patients with lymphoblastic or mixed lymphoblastic-myeloblastic markers, but only one of the six with myeloblastic markers, achieved complete or partial remission in response to therapy. Thus, in acute undifferentiated leukemia, classification of blasts with these markers may be of prognostic value. Of the 13 patients with chronic myelogenous leukemia in blast crises, the markers were concordant (for myeloblasts) in only two cases. Three of the 13 patients had TDT-positive blasts, but the reactions of these cells with anti-i and with anti-myeloblast serum differed from those seen with lymphoblasts from patients with acute lymphoblastic leukemia. Although the cell involved in "lymphoid" blast crisis of chronic myelogenous leukemia is similar in many respects to that involved in acute lymphoblastic leukemia, these cells are not identical.

[Clinical and cytological differences in adult acute lymphatic and acute undifferentiated leukemia].

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Abbrederis, K; Schmalzl, F

1976-01-01

The usefulness for clinical purposes of the distinction of acute undifferentiated (AUL) and acute lymphocytic leukemia (ALL) is suggested by the following observations: 1. Maturation from AUL to ALL has not been observed. Transformation of ALL to AUL has been reported i.e. less of cytoplasmic polysaccharides; however this seems rather to be the effect of cytotoxic therapy and not a real change of the cytological type. 2. Significant differences among ALL and AUL can be noted as far as the therapeutic response is concerned: All of the 9 patients with ALL but only 2 out of 9 patients with AUL went into remission. The mean survival of the cases with ALL amounts to 34, that of AUL only to 4 months. Out of the patients with ALL 4 patients are still alive in persistant first remission after 77, 57, 36 and 28 months. 3. ALL occurs most frequently in young adults (mean age of 21 patients: 31.7 years): AUL is more frequent in elderly patients (Mean age of 18 patients: 57.6 years). 4. In our material ALL did never occur consequent to a typical preluekemic stage, which was followed either by myeloblastic, monocytic, erythroleukemic or undifferentiated leukemias.

Immunophenotypic characterisation and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords during in vitro culture

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Mazurkevych Anatoliy

2016-09-01

Full Text Available Introduction: The objective of the study was immunophenotypic and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords during in vitro culture.

Cytogenetic evidence of metastatic myxoid liposarcoma and therapy-related myelodysplastic syndrome in a bone marrow biopsy.

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Rossi, Sabrina; Canal, Fabio; Licci, Stefano; Zanatta, Lucia; Laurino, Licia; Gottardi, Michele; Gherlinzoni, Filippo; Dei Tos, Angelo Paolo

2009-07-01

Myxoid liposarcoma exhibits a peculiar clinical behavior, with a tendency to spread to serosal membranes, distant soft tissues, and bones, even in the absence of lung metastases. Therapy-related hematological neoplasms are well-known side effects of cytotoxic chemotherapy. We describe an exceptional case of metastatic myxoid liposarcoma of the spine associated with therapy-related refractory anemia with excess of blasts in a 37-year-old woman who underwent multi-agent chemotherapy for a myxoid liposarcoma of the left thigh. Microscopic examination of the bone marrow biopsy revealed dysplastic features, with abnormal localization of immature precursors and micromegakaryocytes, and islands of undifferentiated oval small/medium-size cells, suggestive of acute myeloid leukemia arising in the setting of a myelodysplastic syndrome. Immunohistochemistry was not discriminant. Cytogenetic analyses of bone marrow aspirate disclosed the presence of 2 different rearrangements, subsequently confirmed by fluorescent in situ hybridization and was crucial in making the correct diagnosis.

A Predictive Model to Classify Undifferentiated Fever Cases Based on Twenty-Four-Hour Continuous Tympanic Temperature Recording

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Pradeepa H. Dakappa

2017-01-01

Full Text Available Diagnosis of undifferentiated fever is a major challenging task to the physician which often remains undiagnosed and delays the treatment. The aim of the study was to record and analyze a 24-hour continuous tympanic temperature and evaluate its utility in the diagnosis of undifferentiated fevers. This was an observational study conducted in the Kasturba Medical College and Hospitals, Mangaluru, India. A total of ninety-six (n=96 patients were presented with undifferentiated fever. Their tympanic temperature was recorded continuously for 24 hours. Temperature data were preprocessed and various signal characteristic features were extracted and trained in classification machine learning algorithms using MATLAB software. The quadratic support vector machine algorithm yielded an overall accuracy of 71.9% in differentiating the fevers into four major categories, namely, tuberculosis, intracellular bacterial infections, dengue fever, and noninfectious diseases. The area under ROC curve for tuberculosis, intracellular bacterial infections, dengue fever, and noninfectious diseases was found to be 0.961, 0.801, 0.815, and 0.818, respectively. Good agreement was observed [kappa = 0.618 (p<0.001, 95% CI (0.498–0.737] between the actual diagnosis of cases and the quadratic support vector machine learning algorithm. The 24-hour continuous tympanic temperature recording with supervised machine learning algorithm appears to be a promising noninvasive and reliable diagnostic tool.

Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells

International Nuclear Information System (INIS)

Miyazaki, Takamichi; Futaki, Sugiko; Hasegawa, Kouichi; Kawasaki, Miwa; Sanzen, Noriko; Hayashi, Maria; Kawase, Eihachiro; Sekiguchi, Kiyotoshi; Nakatsuji, Norio; Suemori, Hirofumi

2008-01-01

Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs. We first determined the major integrins expressed on the hESCs to reveal the preference of the hESCs for rhLMs, and found that the hESCs mainly expressed integrin α6β1, which binds predominantly to laminin-111, -332 and -511/-521. When the hESCs were seeded onto rhLMs, the cells indeed adhered markedly to rhLM-332, and to rhLM-511 and rhLM-111 to a lesser extent. The hESCs proliferated on these three rhLMs for several passages while preserving their pluripotency. These results show that rhLM-111, -332, and -511 are good substrates to expand undifferentiated hESCs due to their high affinity to integrin α6β1 expressed on hESCs

Prevalence of bone and soft tissue tumors.

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Yücetürk, Güven; Sabah, Dündar; Keçeci, Burçin; Kara, Ahmet Duran; Yalçinkaya, Selçuk

2011-01-01

Multidisciplinary approach is a necessity for the appropriate diagnosis and treatment of bone and soft tissue tumors. The Ege University Musculoskeletal Tumor Council offers consultation services to other hospitals in the Aegean region. Since 1988 the Council has met weekly and spent approximately 1,500 hours evaluating almost 6,000 patients with suspected skeletal system tumors. Our objective was to present the data obtained from this patient group. A total of 5,658 patients, suspected to have a musculoskeletal tumor, were evaluated retrospectively. Multiple records of the patients due to multiple attendance to the Council were excluded. The prevalance of the bone and soft tissue tumors in these patients were analysed. Malignant mesenchymal tumors accounted for 39.7% of the total patients, benign tumors for 17%, tumor-like lesions for 17.8% and metastatic carsinomas for 8.6%. Malignant bone tumors were 50.2% and malignant soft tissue tumors were 49.8% of all the sarcomas. Among the malignant bone tumors the most common was osteosarcomas at a rate of 33.6%, followed by Ewing-PNET at 25.5%, chondrosarcomas at 19.4% and haematopoietic tumors at 17.6%. Pleomorphic sarcomas (24.5%), liposarcoma (16.4%), synovial sarcoma (13%) and undifferential sarcomas (8.8%) were the most common types of malignant sof tissue tumors. Benign soft tissue tumors (48%), benign cartilage tumors (28%), giant cell tumor (15%) and osteogenic tumors (9%) were found among the benign tumors. Hemangioma, lipoma, agressive fibromatosis, enchondroma, solitary chondroma and osteoid osteoma were the most common tumors in their groups. Lung (27%), breast (24%), gastrointestinal system (10.5%) and kidney (8.2%) carcinomas were the most common primary sites of the bone metastasis. Turkey still lacks a comprehensive series indicating the incidence and diagnostic distribution of bone and soft tissue tumors. The presented data would add to our knowledge on the specific rates of the bone and soft tissue

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

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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.

Bone tissue engineering with a collagen–hydroxyapatite scaffold and culture expanded bone marrow stromal cells

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Villa, Max M.; Wang, Liping; Huang, Jianping; Rowe, David W.; Wei, Mei

2015-01-01

Osteoprogenitor cells combined with supportive biomaterials represent a promising approach to advance the standard of care for bone grafting procedures. However, this approach faces challenges, including inconsistent bone formation, cell survival in the implant, and appropriate biomaterial degradation. We have developed a collagen–hydroxyapatite (HA) scaffold that supports consistent osteogenesis by donor derived osteoprogenitors, and is more easily degraded than a pure ceramic scaffold. Herein, the material properties are characterized as well as cell attachment, viability, and progenitor distribution in vitro. Furthermore, we examined the biological performance in vivo in a critical-size mouse calvarial defect. To aid in the evaluation of the in-house collagen–HA scaffold, the in vivo performance was compared with a commercial collagen–HA scaffold (Healos®, Depuy). The in-house collagen–HA scaffold supported consistent bone formation by predominantly donor-derived osteoblasts, nearly completely filling a 3.5 mm calvarial defect with bone in all samples (n=5) after 3 weeks of implantation. In terms of bone formation and donor cell retention at 3 weeks postimplantation, no statistical difference was found between the in-house and commercial scaffold following quantitative histomorphometry. The collagen–HA scaffold presented here is an open and well-defined platform that supports robust bone formation and should facilitate the further development of collagen–hydroxyapatite biomaterials for bone tissue engineering. PMID:24909953

In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

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Henk-Jan Prins

2014-03-01

Full Text Available One of the applications of bone marrow stromal cells (BMSCs that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing “bulk-cultured” BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

ARG1 Functions in the Physiological Adaptation of Undifferentiated Plant Cells to Spaceflight

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Zupanska, Agata K.; Schultz, Eric R.; Yao, JiQiang; Sng, Natasha J.; Zhou, Mingqi; Callaham, Jordan B.; Ferl, Robert J.; Paul, Anna-Lisa

2017-11-01

Scientific access to spaceflight and especially the International Space Station has revealed that physiological adaptation to spaceflight is accompanied or enabled by changes in gene expression that significantly alter the transcriptome of cells in spaceflight. A wide range of experiments have shown that plant physiological adaptation to spaceflight involves gene expression changes that alter cell wall and other metabolisms. However, while transcriptome profiling aptly illuminates changes in gene expression that accompany spaceflight adaptation, mutation analysis is required to illuminate key elements required for that adaptation. Here we report how transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity 1 (Arg1), a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild-type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding ARG1 (ARG1 KO), both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC-17 spaceflight mission. The cultured cell lines were grown within 60 mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment, with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent, suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight.

MALDI-TOF MS performance compared to direct examination, culture, and 16S rDNA PCR for the rapid diagnosis of bone and joint infections.

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Lallemand, E; Coiffier, G; Arvieux, C; Brillet, E; Guggenbuhl, P; Jolivet-Gougeon, A

2016-05-01

The rapid identification of bacterial species involved in bone and joint infections (BJI) is an important element to optimize the diagnosis and care of patients. The aim of this study was to evaluate the usefulness of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) for the rapid diagnosis of bone infections, directly on synovial fluid (SF) or on crushed osteoarticular samples (CS). From January to October 2013, we prospectively analyzed 111 osteoarticular samples (bone and joint samples, BJS) from 78 patients in care at the University Hospital of Rennes, France. The diagnosis procedure leading to the sample collection was linked to a suspicion of infection, inflammatory disease, arthritis, or for any bone or joint abnormalities. Standard bacteriological diagnosis and molecular biology analysis [16S rRNA polymerase chain reaction (PCR) and sequencing] were conducted. In addition, analysis by MALDI-TOF MS was performed directly on the osteoarticular samples, as soon as the amount allowed. Culture, which remains the gold standard for the diagnosis of BJI, has the highest sensitivity (85.9 %) and remains necessary to test antimicrobial susceptibility. The 16S rDNA PCR results were positive in the group with positive BJI (28.6 %) and negative in the group without infection. Direct examination remains insensitive (31.7 %) but more effective than MALDI-TOF MS directly on the sample (6.3 %). The specificity was 100 % in all cases, except for culture (74.5 %). Bacterial culture remains the gold standard, especially enrichment in blood bottles. Direct analysis of bone samples with MALDI-TOF MS is not useful, possibly due to the low inoculum of BJS.

Cryostored autologous skull bone for cranioplasty? A study on cranial bone flaps' viability and microbial contamination after deep-frozen storage at -80°C.

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Chan, David Yuen Chung; Mok, Yi Tan; Lam, Ping Kuen; Tong, Cindy See Wai; Ng, Stephanie Chi Ping; Sun, Tin Fung David; Poon, Wai Sang

2017-08-01

Craniectomy is a life-saving procedure. Subsequent cranioplasty with autologous skull bone has a bone resorption rate from 4% to 22.8% and an infection rate from 3.3% to 26%. There are concerns with their viability and the potential microbial contamination as they were explanted for a long period of time. Eighteen cranial bone flaps stored at Prince of Wales Hospital Skull Bone Bank during the period from June 2011 to March 2016 were identified. Ethics approval was obtained. Bone chips and deep bone swabs were collected for osteoblast culture and microbial culture. Skull Bone Bank was kept at -80°C under strict aseptic technique during the study period. The storage period ranged from 4months to 55months. For the osteoblast culture, all eighteen bone flaps had no viable osteoblast growth. For the bacterial culture, five had positive bacteria growth (27.8%). Three were Pasteurella multocida and two were Methicillin-resistant Staphylococcus aureus. The mean duration of storage of the infected bone flap was 32.9months (±15.1months) versus 19.9months (±17.9months) of those bone flaps with no bacterial growth (p=0.1716). The mean size of the infected versus non-infected bone flaps was 117.7cm 2 (±44.96cm 2 ) versus 76.8cm 2 (±50.24cm 2 ) respectively (p=0.1318). Although in this study statistical significance was not reached, it was postulated that infected bone flaps tended to be larger in size and had a longer duration of storage. In conclusion, cryostored skull bone flaps beyond four months showed no viable osteoblasts. Bacterial contamination rate of bone flaps was 27.8% in this study. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

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Zhengqi Chang

Full Text Available 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.

Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

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Khwanraj, Kawinthra; Phruksaniyom, Chareerut; Madlah, Suriyat; Dharmasaroja, Permphan

2015-01-01

The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson's disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH) in undifferentiated and retinoic acid- (RA-) induced differentiated cells. The western blot results showed a gradual decre...

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

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Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

2013-07-30

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

Rhabdoid and Undifferentiated Phenotype in Renal Cell Carcinoma: Analysis of 32 Cases Indicating a Distinctive Common Pathway of Dedifferentiation Frequently Associated With SWI/SNF Complex Deficiency.

Science.gov (United States)

Agaimy, Abbas; Cheng, Liang; Egevad, Lars; Feyerabend, Bernd; Hes, Ondřej; Keck, Bastian; Pizzolitto, Stefano; Sioletic, Stefano; Wullich, Bernd; Hartmann, Arndt

2017-02-01

Undifferentiated (anaplastic) and rhabdoid cell features are increasingly recognized as adverse prognostic findings in renal cell carcinoma (RCC), but their molecular pathogenesis has not been studied sufficiently. Recent studies identified alterations in the Switch Sucrose nonfermentable (SWI/SNF) chromatin remodeling complex as molecular mechanisms underlying dedifferentiation and rhabdoid features in carcinomas of different organs. We herein have analyzed 32 undifferentiated RCCs having in common an undifferentiated (anaplastic) phenotype, prominent rhabdoid features, or both, irrespective of the presence or absence of conventional RCC component. Cases were stained with 6 SWI/SNF pathway members (SMARCB1, SMARCA2, SMARCA4, ARID1A, SMARCC1, and SMARCC2) in addition to conventional RCC markers. Patients were 20 males and 12 females aged 32 to 85 years (mean, 59). A total of 22/27 patients with known stage presented with ≥pT3. A differentiated component varying from microscopic to major component was detected in 20/32 cases (16 clear cell and 2 cases each chromophobe and papillary RCC). The undifferentiated component varied from rhabdoid dyscohesive cells to large epithelioid to small monotonous anaplastic cells. Variable loss of at least 1 SWI/SNF complex subunit was noted in the undifferentiated/rhabdoid component of 21/32 cases (65%) compared with intact or reduced expression in the differentiated component. A total of 15/17 patients (88%) with follow-up died of metastatic disease (mostly within 1 y). Only 2 patients were disease free at last follow-up (1 and 6 y). No difference in survival, age distribution, or sex was observed between the SWI/SNF-deficient and the SWI/SNF-intact group. This is the first study exploring the role of SWI/SNF deficiency as a potential mechanism underlying undifferentiated and rhabdoid phenotype in RCC. Our results highlight the association between the aggressive rhabdoid phenotype and the SWI/SNF complex deficiency, consistent

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2

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Jung-Seok Lee

2018-04-01

Full Text Available This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2 treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis, and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Keywords: Bone marrow stromal cells, Hypoxia, Fibroblast growth factor, Tissue regeneration, Microenvironment interactions

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

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Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

2010-07-01

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

Experience with a bone bank operation and allograft bone infection in recipients at a medical centre in southern Taiwan.

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Liu, J W; Chao, L H; Su, L H; Wang, J W; Wang, C J

2002-04-01

To assess the contamination rate of allograft bones at retrieval and the infection rate of the implanted allograft bone, we audited a bone bank retrospectively and reviewed the medical charts of allograft bone recipients between June 1999 and June 2000 at a medical centre in southern Taiwan. The bone bank did its utmost to minimize allograft contamination with hospital-acquired pathogens by adopting purposefully designed criteria for selection of donors. This protocol included sterilization with soaking of the retrieved allograft in a solution of a first-generation cephalosporin before storage and prophylaxis in recipients with first-generation cephalosporin. The contamination rates at allograft retrieval from living and cadaveric donors were 2.7% and 12.4%, respectively (P<0.001). Culture of 262 specimens taken at allograft implant revealed 12 (4.6%) positive for culture. Of the 12 patients implanted with allograft bones positive for culture, nine (75.0%) had allograft bone infection, while three (25.0%) did not. Among the 250 recipients with sterile allograft bones, four (1.6%) were found to have allograft infection. None of the cases of infection required removal of the allograft bones, and all cases were successfully treated with tailored antimicrobial therapy based on susceptibility tests on isolated bacteria. The overall infection rate was 5.0%, which compared favourably with those in other series. A prospective cohort study is needed to determine which of the varied sterilization methodologies gives the best and/or most cost-effective outcome. Copyright 2002 The Hospital Infection Society.

Heterogeneity in acute undifferentiated leukemia.

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LeMaistre, A; Childs, C C; Hirsch-Ginsberg, C; Reuben, J; Cork, A; Trujillo, J M; Andersson, B; McCredie, K B; Freireich, E; Stass, S A

1988-01-01

From January 1985 to May 1987, we studied 256 adults with newly diagnosed acute leukemia. Acute undifferentiated leukemia (AUL) was diagnosed in 12 of the 256 (4.6%) cases when lineage could not be delineated by light microscopy and light cytochemistry. To further characterize the blasts, immunophenotyping, ultrastructural myeloperoxidase (UMPO), and ultrastructural platelet peroxidase parameters were examined in 10, 11, and 6 of the 12 cases, respectively. Five cases demonstrated UMPO and were reclassified as acute myeloblastic leukemia (AML). Of the six UMPO-negative cases, three had a myeloid and one had a mixed immunophenotype. One UMPO-negative patient with a myeloid immunophenotype was probed for the immunoglobulin heavy chain gene (JH) and the beta chain of the T-cell receptor gene (Tcr beta) with no evidence of rearrangement. Six cases were treated with standard acute lymphoblastic leukemia (ALL) chemotherapy and failed to achieve complete remission (CR). Various AML chemotherapeutic regimens produced CR in only 3 of the 12 cases. One case was treated with gamma interferon and the other 2 with high-dose Ara-C. Our findings indicate a myeloid lineage can be detected by UMPO (5/12) in some cases of AUL. A germline configuration with JH and Tcr beta in one case as well as a myeloid immunophenotype in 3 UMPO-negative cases raises the possibility that myeloid lineage commitment may occur in the absence of myeloid peroxidase (MPO) cytochemical positivity.

The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

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Emma L Smith

Full Text Available Transforming growth factor-beta3 (TGF-β3 and 1α,25-dihydroxyvitamin D3 (1α,25 (OH 2D3 are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11 were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH 2D3 (25 nM or TGF-β3 (5 ng/mL & 15 ng/mL. Analyses of the femurs were undertaken using micro-computed tomography (μCT, histology and immunohistochemistry. 1α,25 (OH2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

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El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

2018-02-01

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

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El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

2002-01-01

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key

Hake fish bone as a calcium source for efficient bone mineralization.

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Flammini, Lisa; Martuzzi, Francesca; Vivo, Valentina; Ghirri, Alessia; Salomi, Enrico; Bignetti, Enrico; Barocelli, Elisabetta

2016-01-01

Calcium is recognized as an essential nutritional factor for bone health. An adequate intake is important to achieve or maintain optimal bone mass in particular during growth and old age. The aim of the present study was to evaluate the efficiency of hake fish bone (HBF) as a calcium source for bone mineralization: in vitro on osteosarcoma SaOS-2 cells, cultured in Ca-free osteogenic medium (OM) and in vivo on young growing rats fed a low-calcium diet. Lithotame (L), a Ca supplement derived from Lithothamnium calcareum, was used as control. In vitro experiments showed that HBF supplementation provided bone mineralization similar to standard OM, whereas L supplementation showed lower activity. In vivo low-Ca HBF-added and L-added diet similarly affected bone deposition. Physico-chemical parameters concerning bone mineralization, such as femur breaking force, tibia density and calcium/phosphorus mineral content, had beneficial effects from both Ca supplementations, in the absence of any evident adverse effect. We conclude HBF derived from by-product from the fish industry is a good calcium supplier with comparable efficacy to L.

Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures

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Róza Zákány

2013-08-01

Full Text Available Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2 were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models.

Total Artificial Heart Implantation After Undifferentiated High-Grade Sarcoma Excision.

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Kremer, Jamila; Farag, Mina; Arif, Rawa; Brcic, Andreas; Sabashnikov, Anton; Schmack, Bastian; Popov, Aron-Frederik; Karck, Matthias; Dohmen, Pascal M; Ruhparwar, Arjang; Weymann, Alexander

2016-11-02

BACKGROUND Total artificial heart (TAH) implantation in patients with aggressive tumor infiltration of the heart can be challenging. CASE REPORT We report on a patient with a rare primary undifferentiated high-grade spindle cell sarcoma of the mitral valve and in the left atrium, first diagnosed in 2014. The referring center did a first resection in 2014. In the course of 17 months, computer tomography (CT) scan again showed massive invasion of the mitral valve and left atrium. Partial resection and mitral valve replacement was not an option. We did a subtotal heart excision with total artificial heart implantation. In this report we discuss complications, risk factors, and perioperative management of this patient. CONCLUSIONS Patients with aggressive tumors of the heart can be considered for TAH implantation.

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

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Christopher A Smith

Full Text Available To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC. BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1 concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

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Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Clay-Enriched Silk Biomaterials for Bone Formation

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Mieszawska, Aneta J.; Llamas, Jabier Gallego; Vaiana, Christopher A.; Kadakia, Madhavi P.; Naik, Rajesh R.; Kaplan, David L.

2011-01-01

The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone specific uses. Clay montmorillonite (Cloisite ® Na+) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared to silk films doped with sodium silicate as controls for support of human bone marrow derived mesenchymal stem cells (hMSCs) in osteogenic culture. The cells adhered and proliferated on the silk/clay composites over two weeks. Quantitative real-time RT-PCR analysis revealed increased transcript levels for alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col I) osteogenic markers in the cells cultured on the silk/clay films in comparison to the controls. Early evidence for bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that the silk/clay composite systems may be useful for further study toward bone regenerative needs. PMID:21549864

DLK1 is a novel regulator of bone mass that mediates estrogen deficiency-induced bone loss in mice

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Abdallah, Basem M; Ditzel, Nicholas; Mahmood, Amer

2011-01-01

. In a number of in vitro culture systems, Dlk1 stimulated osteoclastogenesis indirectly through osteoblast-dependent increased production of proinflammatory bone-resorbing cytokines (eg, Il7, Tnfa, and Ccl3). We found that ovariectomy (ovx)-induced bone loss was associated with increased production of Dlk1...... in the bone marrow by activated T cells. Interestingly, Dlk1(-/-) mice were significantly protected from ovx-induced bone loss compared with wild-type mice. Thus we identified Dlk1 as a novel regulator of bone mass that functions to inhibit bone formation and to stimulate bone resorption. Increasing DLK1...... production by T cells under estrogen deficiency suggests its possible use as a therapeutic target for preventing postmenopausal bone loss....

Study of tissue engineered bone nodules by Fourier transform infrared spectroscopy.

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Aydin, Halil Murat; Hu, Bin; Suso, Josep Sulé; El Haj, Alicia; Yang, Ying

2011-02-21

The key criteria for assessing the success of bone tissue engineering are the quality and quantity of the produced minerals within the cultured constructs. The accumulation of calcium ions and inorganic phosphates in culture medium serves as nucleating agents for the formation of hydroxyapatite, which is the main inorganic component of bone. Bone nodule formation is one of the hallmarks of mineralization in such cell cultures. In this study, we developed a new two-step procedure to accelerate bone formation in which mouse bone cell aggregates were produced first on various chemically treated non-adhesive substrates. After this step, the bone cells' growth and mineralization were followed in conventional culture plates. The number and size of cell aggregates were studied with light microscopy. The minerals' formation in the form of nodules produced by the cell aggregates and the bone crystal quality were studied with Fourier Transform Infrared (FTIR) spectroscopy. The FTIR spectra of the ash specimens (mineral phase only) from thermal gravimetric analysis (TGA) provided valuable information of the quality of the minerals. The υ(4) PO(4) region (550-650 cm(-1)), which reveals apatitic and non-apatitic HPO(4) or PO(4) environments, and phosphate region (910-1180 cm(-1)) were examined for the minerals produced in the form of nodules. The peak position and intensity of the spectra demonstrate that the quality of the bone produced by cell aggregates, especially from the bigger ones, which were formed on Plunoric treated substrates, exhibit a composition more similar to that of native bone. This work establishes a new protocol for high quality bone formation and characterization, with the potential to be applied to bone tissue engineering.

Down-regulation of parathyroid hormone (PTH) receptors in cultured bone cells is associated with agonist-specific intracellular processing of PTH-receptor complexes.

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Teitelbaum, A P; Silve, C M; Nyiredy, K O; Arnaud, C D

1986-02-01

Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down-regulation in cultured bone cells is mediated by cellular internalization of PTH-receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid-resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1-34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH-induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest

Undifferentiated pleomorphic sarcoma: indolent, tail-like recurrence of a high-grade tumor

Energy Technology Data Exchange (ETDEWEB)

Alpert, Justin S. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Boland, Patrick [Memorial Sloan Kettering Cancer Center, Division of Orthopaedic Surgery, Department of Surgery, New York, NY (United States); Weill Medical College of Cornell University, New York, NY (United States); Hameed, Meera [Memorial Sloan Kettering Cancer Center, Department of Pathology, New York, NY (United States); Panicek, David M. [Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, NY (United States); Weill Medical College of Cornell University, New York, NY (United States)

2018-01-15

Recurrence of a soft tissue sarcoma typically manifests as a round or oval mass at imaging, and recurrent high-grade soft tissue sarcomas generally enlarge relatively rapidly. We present a case of high-grade undifferentiated pleomorphic sarcoma in the calf of a 48-year-old male that recurred as a thin, curvilinear ''tail'' of enhancing tissue at magnetic resonance imaging (MRI), with extremely indolent growth over a 7-year period. The unusual imaging finding of a slowly enlarging ''tail'' should not be dismissed as postoperative changes, even for a high-grade soft tissue sarcoma. (orig.)

Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

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Takuya Kuroda

Full Text Available Human induced pluripotent stem cells (hiPSCs possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs. These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay: soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR. Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10⁴ RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

Tumor-targeting Salmonella typhimurium A1-R is a highly effective general therapeutic for undifferentiated soft tissue sarcoma patient-derived orthotopic xenograft nude-mouse models.

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Igarashi, Kentaro; Kawaguchi, Kei; Kiyuna, Tasuku; Miyake, Kentaro; Miyake, Masuyo; Singh, Arun S; Eckardt, Mark A; Nelson, Scott D; Russell, Tara A; Dry, Sarah M; Li, Yunfeng; Yamamoto, Norio; Hayashi, Katsuhiro; Kimura, Hiroaki; Miwa, Shinji; Tsuchiya, Hiroyuki; Singh, Shree Ram; Eilber, Fritz C; Hoffman, Robert M

2018-03-18

Undifferentiated soft tissue sarcoma (USTS) is a recalcitrant and heterogeneous subgroup of soft tissue sarcoma with high risk of metastasis and recurrence. Due to heterogeneity of USTS, there is no reliably effective first-line therapy. We have generated tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R), which previously showed strong efficacy on single patient-derived orthotopic xenograft (PDOX) models of Ewing's sarcoma and follicular dendritic cell sarcoma. In the present study, tumor resected from 4 patients with a biopsy-proven USTS (2 undifferentiated pleomorphic sarcoma [UPS], 1 undifferentiated sarcoma not otherwise specified [NOS] and 1 undifferentiated spindle cell sarcoma [USS]) were grown orthotopically in the biceps femoris muscle of mice to establish PDOX models. One USS model and one UPS model were doxorubicin (DOX) resistant. One UPS and the NOS model were partially sensitive to DOX. DOX is first-line therapy for these diseases. S. typhimurium A1-R arrested tumor growth all 4 models. In addition to arresting tumor growth in each case, S. typhimurium A1-R was significantly more efficacious than DOX in each case, thereby surpassing first-line therapy. These results suggest that S. typhimurium A1-R can be a general therapeutic for USTS and possibly sarcoma in general. Published by Elsevier Inc.

Fabrication and characterization of PDLLA/pyrite composite bone ...

Indian Academy of Sciences (India)

Keywords. Polylactic acid; Chinese herbal medicine; pyrite; scaffold; bone regeneration; cell culture. ... Pyrite (FeS2, named as Zi-Ran-Tong in Chinese medicine), as a traditional Chinesemedicine, has been used in the Chinese population to treat bone diseases and to promote bone healing. The mechanical properties of ...

The problem of gastroptosis as a manifestation of undifferentiated connective tissue dysplasia in the clinical practice of pediatric gastroenterologist

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O.M. Shulhai

2018-04-01

Full Text Available In the article, the authors describe a clinical case of undifferentiated connective tissue dysplasia in 10- and 15-year-old girls. This pathology is common because it has a lot of clinical, morphological and visceral manifestations, but it is hard to diagnose. Many chronic diseases have been formed based on this pathology. Clinical cases in this article describe confirmed gastroptosis (greater curvature of the stomach is displaced downwards, below the level of the iliac crests in standing position as one of the visceral manifestations of undifferentiated connective tissue dysplasia, laboratory and instrumental findings that help to diagnose this syndrome. Gastroptosis occurs in children with asthenic type of constitution (elongated limbs, thin body, small chest, narrow shoulders, hypermobility of the joints. It comes from weak development of muscle and connective tissues so they can not endure overload, resulting is many problems, including the gastroptosis, visceroptosis etc. There are many causes of gastroptosis: congenital anomalies of the ligamentous apparatus structure, maternal disease during pregnancy, surgical intervention, sharp decrease in body weight, vitamin and proteins deficiency, irrational nutrition, lengthening the mesentery of an organ such as large intestine. If we know clinical manifestations and features of undifferentiated connective tissue dysplasia, it will allow diagnosing this pathology in a timely manner and will help more fully provide medical care to such patients, carry out their rehabilitation, psychological ada­ptation, and prevent early development of disability.

In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

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Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T

2003-06-13

Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.

Pressure sores and underlying bone infection

International Nuclear Information System (INIS)

Sugarman, B.

1987-01-01

Pressure sores are a serious complication of hospitalized and chronically ill patients. Evaluation for underlying bone infection can be made difficult by radiographic, nuclear imaging, and soft-tissue culture studies that are abnormal and suggest the presence of bone infection, when no infection is present. Evaluation by bone biopsy with histologic and microbiological studies can accurately and promptly diagnose whether bone infection is present. This allows appropriate treatment when infection is present, and prevents unneeded and potentially toxic antibiotic therapy when preliminary studies incorrectly suggest that infection is present

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

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Ghourichaee, Sasan Sharee; Powell, Elizabeth M; Leach, Jennie B

2017-05-01

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O 2 ) versus hypoxic (3% O 2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Engineering bone grafts with enhanced bone marrow and native scaffolds.

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Hung, Ben P; Salter, Erin K; Temple, Josh; Mundinger, Gerhard S; Brown, Emile N; Brazio, Philip; Rodriguez, Eduardo D; Grayson, Warren L

2013-01-01

The translation of tissue engineering approaches to the clinic has been hampered by the inability to find suitable multipotent cell sources requiring minimal in vitro expansion. Enhanced bone marrow (eBM), which is obtained by reaming long bone medullary canals and isolating the solid marrow putty, has large quantities of stem cells and demonstrates significant potential to regenerate bone tissues. eBM, however, cannot impart immediate load-bearing mechanical integrity or maintain the gross anatomical structure to guide bone healing. Yet, its putty-like consistency creates a challenge for obtaining the uniform seeding necessary to effectively combine it with porous scaffolds. In this study, we examined the potential for combining eBM with mechanically strong, osteoinductive trabecular bone scaffolds for bone regeneration by creating channels into scaffolds for seeding the eBM. eBM was extracted from the femurs of adult Yorkshire pigs using a Synthes reamer-irrigator-aspirator device, analyzed histologically, and digested to extract cells and characterize their differentiation potential. To evaluate bone tissue formation, eBM was seeded into the channels in collagen-coated or noncoated scaffolds, cultured in osteogenic conditions for 4 weeks, harvested and assessed for tissue distribution and bone formation. Our data demonstrates that eBM is a heterogenous tissue containing multipotent cell populations. Furthermore, coating scaffolds with a collagen hydrogel significantly enhanced cellular migration, promoted uniform tissue development and increased bone mineral deposition. These findings suggest the potential for generating customized autologous bone grafts for treating critical-sized bone defects by combining a readily available eBM cell source with decellularized trabecular bone scaffolds. © 2013 S. Karger AG, Basel

A cellular automata model of bone formation.

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Van Scoy, Gabrielle K; George, Estee L; Opoku Asantewaa, Flora; Kerns, Lucy; Saunders, Marnie M; Prieto-Langarica, Alicia

2017-04-01

Bone remodeling is an elegantly orchestrated process by which osteocytes, osteoblasts and osteoclasts function as a syncytium to maintain or modify bone. On the microscopic level, bone consists of cells that create, destroy and monitor the bone matrix. These cells interact in a coordinated manner to maintain a tightly regulated homeostasis. It is this regulation that is responsible for the observed increase in bone gain in the dominant arm of a tennis player and the observed increase in bone loss associated with spaceflight and osteoporosis. The manner in which these cells interact to bring about a change in bone quality and quantity has yet to be fully elucidated. But efforts to understand the multicellular complexity can ultimately lead to eradication of metabolic bone diseases such as osteoporosis and improved implant longevity. Experimentally validated mathematical models that simulate functional activity and offer eventual predictive capabilities offer tremendous potential in understanding multicellular bone remodeling. Here we undertake the initial challenge to develop a mathematical model of bone formation validated with in vitro data obtained from osteoblastic bone cells induced to mineralize and quantified at 26 days of culture. A cellular automata model was constructed to simulate the in vitro characterization. Permutation tests were performed to compare the distribution of the mineralization in the cultures and the distribution of the mineralization in the mathematical models. The results of the permutation test show the distribution of mineralization from the characterization and mathematical model come from the same probability distribution, therefore validating the cellular automata model. Copyright © 2017 Elsevier Inc. All rights reserved.

[Undifferentiated (embryonal) liver sarcoma: reviaew of 6 cases in National Cancer Institute, Lima, Peru. Review of the literature].

Science.gov (United States)

Dueñas, Daniela; Huanca, Lourdes; Cordero, Mónica; Webb, Patricia; Ruiz, Eloy

2016-01-01

Undifferentiated (embryonal) liver sarcoma is a rare tumor about 2% of all malignant liver tumors with a poor prognosis and usually occurs in children, this review aims to assess cases of primary embryonal sarcoma of the liver presented at our institution the past 8 years and improve recognition of its variants and evaluate immunohistochemical characteristics that help differentiated it from other tumors. Six cases of undifferentiated liver sarcoma were histologically evaluated and investigated by immunohistochemistry with a panel of antibodies using the equipment “Autostainer Link 48”. Usually masses were on average more than 20 cm, with solid, cystic, mucinous areas. The microscopic features include cells of spindle cell appearance, oval, starry, epithelioid and multinucleated cells densely arranged in a myxoid matrix. Trapped bile ducts and hepatic cords often present in the periphery of tumors. Intracellular and extracellular PAS positive hyaline globules. Immunohistochemistry showed very divergent differentiation.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture.

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Cerejo, Sofia de Amorim; Rahal, Sheila Canevese; Lima Neto, João Ferreira de; Voorwald, Fabiana Azevedo; Alvarenga, Fernanda da Cruz Landim e

2011-10-01

To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

Rapid prototyped porous nickel–titanium scaffolds as bone substitutes

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Waldemar Hoffmann

2014-06-01

Full Text Available While calcium phosphate–based ceramics are currently the most widely used materials in bone repair, they generally lack tensile strength for initial load bearing. Bulk titanium is the gold standard of metallic implant materials, but does not match the mechanical properties of the surrounding bone, potentially leading to problems of fixation and bone resorption. As an alternative, nickel–titanium alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone better than any other metallic material. With the ultimate goal of fabricating porous implants for spinal, orthopedic and dental applications, nickel–titanium substrates were fabricated by means of selective laser melting. The response of human mesenchymal stromal cells to the nickel–titanium substrates was compared to mesenchymal stromal cells cultured on clinically used titanium. Selective laser melted titanium as well as surface-treated nickel–titanium and titanium served as controls. Mesenchymal stromal cells had similar proliferation rates when cultured on selective laser melted nickel–titanium, clinically used titanium, or controls. Osteogenic differentiation was similar for mesenchymal stromal cells cultured on the selected materials, as indicated by similar gene expression levels of bone sialoprotein and osteocalcin. Mesenchymal stromal cells seeded and cultured on porous three-dimensional selective laser melted nickel–titanium scaffolds homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold’s pore volume with extracellular matrix. The combination of bone-related mechanical properties of selective laser melted nickel–titanium with its cytocompatibility and support of osteogenic differentiation of mesenchymal stromal cells highlights its potential as a superior bone substitute as compared to clinically used

Perfluorodecalin and bone regeneration

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F Tamimi

2013-01-01

Full Text Available Perfluorodecalin (PFD is a chemically and biologically inert biomaterial and, as many perfluorocarbons, is also hydrophobic, radiopaque and has a high solute capacity for gases such as oxygen. In this article we have demonstrated, both in vitro and in vivo, that PFD may significantly enhance bone regeneration. Firstly, the potential benefit of PFD was demonstrated by prolonging the survival of bone marrow cells cultured in anaerobic conditions. These findings translated in vivo, where PFD incorporated into bone-marrow-loaded 3D-printed scaffolds substantially improved their capacity to regenerate bone. Secondly, in addition to biological applications, we have also shown that PFD improves the radiopacity of bone regeneration biomaterials, a key feature required for the visualisation of biomaterials during and after surgical implantation. Finally, we have shown how the extreme hydrophobicity of PFD enables the fabrication of highly cohesive self-setting injectable biomaterials for bone regeneration. In conclusion, perfluorocarbons would appear to be highly beneficial additives to a number of regenerative biomaterials, especially those for bone regeneration.

High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber.

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Tashiro, Shota; Le, Minh Nguyen Tuyet; Kusama, Yuta; Nakatani, Eri; Suga, Mika; Furue, Miho K; Satoh, Taku; Sugiura, Shinji; Kanamori, Toshiyuki; Ohnuma, Kiyoshi

2018-04-19

Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

PDGFRa amplification in multiple skin lesions of undifferentiated pleomorphic sarcoma: A clue for intimal sarcoma metastases.

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Osio, Amélie; Vignon-Pennamen, Marie-Dominique; Pedeutour, Florence; Le Maignan, Christine; Koskas, Fabien; Lebbé, Célèste; Janin, Anne; Battistella, Maxime

2017-05-01

A 62-year-old human immunodeficiency virus-positive man was admitted for multiple cutaneous and subcutaneous nodules on his lower limbs, corresponding to an undifferentiated proliferation of spindle and pleomorphic cells, with irregular nuclei and numerous mitoses. The tumor cells were negative for a large panel of immunohistochemical markers, except CD10. MDM2 immunohistochemical staining was also negative, leading to the diagnosis of Fédération Nationale des Centres de Lutte contre le Cancer grade III undifferentiated pleomorphic sarcoma (UPS). Array-comparative genomic hybridization showed a highly complex karyotype, with amplification of the 4q12 region, an area that contains only the platelet-derived growth factor receptor α (PDGFRa) gene. This amplification of PDFGRa, molecular hallmark of intimal sarcoma (IS), led to the diagnosis of skin IS metastasis. A positron emission tomography showed a hypermetabolic mass protruding in the preaortic area, consistent with the diagnosis of aortic IS. Our study shows that a rare differential diagnosis in peripheral UPS can be IS skin metastasis, and underlines the importance of molecular analyses in UPS. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The use of total human bone marrow fraction in a direct three-dimensional expansion approach for bone tissue engineering applications: focus on angiogenesis and osteogenesis.

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Guerrero, Julien; Oliveira, Hugo; Catros, Sylvain; Siadous, Robin; Derkaoui, Sidi-Mohammed; Bareille, Reine; Letourneur, Didier; Amédée, Joëlle

2015-03-01

Current approaches in bone tissue engineering have shown limited success, mostly owing to insufficient vascularization of the construct. A common approach consists of co-culture of endothelial cells and osteoblastic cells. This strategy uses cells from different sources and differentiation states, thus increasing the complexity upstream of a clinical application. The source of reparative cells is paramount for the success of bone tissue engineering applications. In this context, stem cells obtained from human bone marrow hold much promise. Here, we analyzed the potential of human whole bone marrow cells directly expanded in a three-dimensional (3D) polymer matrix and focused on the further characterization of this heterogeneous population and on their ability to promote angiogenesis and osteogenesis, both in vitro and in vivo, in a subcutaneous model. Cellular aggregates were formed within 24 h and over the 12-day culture period expressed endothelial and bone-specific markers and a specific junctional protein. Ectopic implantation of the tissue-engineered constructs revealed osteoid tissue and vessel formation both at the periphery and within the implant. This work sheds light on the potential clinical use of human whole bone marrow for bone regeneration strategies, focusing on a simplified approach to develop a direct 3D culture without two-dimensional isolation or expansion.

Pleomorphic malignant fibrous histiocytoma/undifferentiated high-grade pleomorphic sarcoma of the scrotum in a patient presenting as fournier gangrene: a case report.

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Guo, Juan; Zhou, Shengmei; Rao, Nagesh P; Pez, Gholam H

2010-10-01

Pleomorphic malignant fibrous histiocytoma (MFH), also known as undifferentiated high-grade pleomorphic sarcoma according to the latest World Health Organization classification, is a diagnosis of exclusion and extremely rare in adult scrotal/paratesticular region. Clinical presentation of scrotal/paratesticular pleomorphic MFH is usually a painless and gradual scrotal swelling. We report a case of scrotal MFH in a 63-year-old man who presented as Fournier gangrene after 10-month painful scrotal swelling and multiple procedures. The specimen of emergent debridement was submitted for pathologic and bacteriologic examination. Microscopically, the lesion had marked architectural and cytologic pleomorphism. The neoplastic cells were positive for vimentin, but negative for all lineage-specific markers. Fluorescence in-situ hybridization showed an aneuploid karyotype and negative results for lipomatous tumor abnormalities. Bacterial cultures of the specimen showed extensive growth of virulent polymicrobes. The diagnosis of scrotal/paratesticular pleomorphic MFH with concurrent Fournier gangrene was made. Thoracic computed tomography scan showed bilateral multiple pulmonary nodules. The patient died 1 month later.

Indistinguishable genomic profiles and shared prognostic markers in undifferentiated pleomorphic sarcoma and leiomyosarcoma: different sides of a single coin?

DEFF Research Database (Denmark)

Carneiro, Ana; Francis, Princy; Bendahl, Pär-Ola

2009-01-01

Soft tissue sarcoma (STS) diagnostics and prognostics are challenging, particularly in highly malignant and pleomorphic subtypes such as undifferentiated pleomorphic sarcoma (UPS) and leiomyosarcoma (LMS). We applied 32K BAC arrays and gene expression profiling to 18 extremity soft tissue LMS and...

Bone infection in patients suspected of complicating osteomyelitis: the diagnostic value of dual isotope bone-granulocyte scintigraphy

DEFF Research Database (Denmark)

Buhl, Thora; Stentzer, Kim; Hede, Adam

2005-01-01

: Simultaneous dual isotope bone-granulocyte scintigraphic images were obtained in 42 consecutive patients in whom conventional X-ray, erythrocyte sedimentation rate, and C-reactive protein were also available. 99mTc MDP bone and 111In labelled granulocyte imaging was obtained simultaneously. The images were...... interpreted as positive for osteomyelitis if regions of interests of pathologic 111In granulocyte accumulation included 99mTc MDP activity on the bone images (except in the spine). RESULTS: The sensitivity, specificity, and accuracy were 84, 71 and 79%, respectively, for simultaneous, dual isotope bone......AIM: The purpose of this study was to evaluate the diagnostic value of dual isotope bone-granulocyte scintigraphy in patients with known bone pathology clinically suspected of osteomyelitis, i.e. complicating osteomyelitis, using per-operative bacterial culture from bone as reference. METHODS...

[Bone marrow stromal damage mediated by immune response activity].

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Vojinović, J; Kamenov, B; Najman, S; Branković, Lj; Dimitrijević, H

1994-01-01

The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis.

Differentiating benign from malignant bone tumors using fluid-fluid level features on magnetic resonance imaging

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Yu, Hong; Cui, Jian Ling; Cui, Sheng Jie; Sun, Ying Cal; Cui, Feng Zhen [Dept. of Radiology, The Third Hospital of Hebei Medical University, Hebei Province Biomechanical Key Laborary of Orthopedics, Shijiazhuang, Hebei (China)

2014-12-15

To analyze different fluid-fluid level features between benign and malignant bone tumors on magnetic resonance imaging (MRI). This study was approved by the hospital ethics committee. We retrospectively analyzed 47 patients diagnosed with benign (n = 29) or malignant (n = 18) bone tumors demonstrated by biopsy/surgical resection and who showed the intratumoral fluid-fluid level on pre-surgical MRI. The maximum length of the largest fluid-fluid level and the ratio of the maximum length of the largest fluid-fluid level to the maximum length of a bone tumor in the sagittal plane were investigated for use in distinguishing benign from malignant tumors using the Mann-Whitney U-test and a receiver operating characteristic (ROC) analysis. Fluid-fluid level was categorized by quantity (multiple vs. single fluid-fluid level) and by T1-weighted image signal pattern (high/low, low/high, and undifferentiated), and the findings were compared between the benign and malignant groups using the chi2 test. The ratio of the maximum length of the largest fluid-fluid level to the maximum length of bone tumors in the sagittal plane that allowed statistically significant differentiation between benign and malignant bone tumors had an area under the ROC curve of 0.758 (95% confidence interval, 0.616-0.899). A cutoff value of 41.5% (higher value suggests a benign tumor) had sensitivity of 73% and specificity of 83%. The ratio of the maximum length of the largest fluid-fluid level to the maximum length of a bone tumor in the sagittal plane may be useful to differentiate benign from malignant bone tumors.

Differentiating benign from malignant bone tumors using fluid-fluid level features on magnetic resonance imaging

International Nuclear Information System (INIS)

Yu, Hong; Cui, Jian Ling; Cui, Sheng Jie; Sun, Ying Cal; Cui, Feng Zhen

2014-01-01

To analyze different fluid-fluid level features between benign and malignant bone tumors on magnetic resonance imaging (MRI). This study was approved by the hospital ethics committee. We retrospectively analyzed 47 patients diagnosed with benign (n = 29) or malignant (n = 18) bone tumors demonstrated by biopsy/surgical resection and who showed the intratumoral fluid-fluid level on pre-surgical MRI. The maximum length of the largest fluid-fluid level and the ratio of the maximum length of the largest fluid-fluid level to the maximum length of a bone tumor in the sagittal plane were investigated for use in distinguishing benign from malignant tumors using the Mann-Whitney U-test and a receiver operating characteristic (ROC) analysis. Fluid-fluid level was categorized by quantity (multiple vs. single fluid-fluid level) and by T1-weighted image signal pattern (high/low, low/high, and undifferentiated), and the findings were compared between the benign and malignant groups using the chi2 test. The ratio of the maximum length of the largest fluid-fluid level to the maximum length of bone tumors in the sagittal plane that allowed statistically significant differentiation between benign and malignant bone tumors had an area under the ROC curve of 0.758 (95% confidence interval, 0.616-0.899). A cutoff value of 41.5% (higher value suggests a benign tumor) had sensitivity of 73% and specificity of 83%. The ratio of the maximum length of the largest fluid-fluid level to the maximum length of a bone tumor in the sagittal plane may be useful to differentiate benign from malignant bone tumors.

Fabrication and characterization of PDLLA/pyrite composite bone ...

Indian Academy of Sciences (India)

Polylactic acid; Chinese herbal medicine; pyrite; scaffold; bone regeneration; cell culture. 1. Introduction ... research focuses on the direct cellular level effect of pyrite on bone cells. ..... optimal scaffold from the results of this paper. Although the.

Tissue-engineered bone constructed in a bioreactor for repairing critical-sized bone defects in sheep.

Science.gov (United States)

Li, Deqiang; Li, Ming; Liu, Peilai; Zhang, Yuankai; Lu, Jianxi; Li, Jianmin

2014-11-01

Repair of bone defects, particularly critical-sized bone defects, is a considerable challenge in orthopaedics. Tissue-engineered bones provide an effective approach. However, previous studies mainly focused on the repair of bone defects in small animals. For better clinical application, repairing critical-sized bone defects in large animals must be studied. This study investigated the effect of a tissue-engineered bone for repairing critical-sized bone defect in sheep. A tissue-engineered bone was constructed by culturing bone marrow mesenchymal-stem-cell-derived osteoblast cells seeded in a porous β-tricalcium phosphate ceramic (β-TCP) scaffold in a perfusion bioreactor. A critical-sized bone defect in sheep was repaired with the tissue-engineered bone. At the eighth and 16th week after the implantation of the tissue-engineered bone, X-ray examination and histological analysis were performed to evaluate the defect. The bone defect with only the β-TCP scaffold served as the control. X-ray showed that the bone defect was successfully repaired 16 weeks after implantation of the tissue-engineered bone; histological sections showed that a sufficient volume of new bones formed in β-TCP 16 weeks after implantation. Eight and 16 weeks after implantation, the volume of new bones that formed in the tissue-engineered bone group was more than that in the β-TCP scaffold group (P bone improved osteogenesis in vivo and enhanced the ability to repair critical-sized bone defects in large animals.

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.

Science.gov (United States)

Aquino-Martínez, Rubén; Angelo, Alcira P; Pujol, Francesc Ventura

2017-11-16

Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca 2+ -containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO 4 ) on MSC migration. In addition, to evaluate the influence of CaSO 4 on MSC differentiation and the potential molecular mechanisms involved. A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO 4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO 4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO 4 treatment was also evaluated by qPCR. CaSO 4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO 4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO 4 -containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO 4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO 4 effects on MSC migration. Specific CaSO 4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO 4 regulates BMP-2-induced MSC migration by differentially activating the PI3

Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining

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Kristian Kjærgaard

2016-01-01

Full Text Available Background. Scaffolds for bone tissue engineering (BTE can be loaded with stem and progenitor cells (SPC from different sources to improve osteogenesis. SPC can be found in bone marrow, adipose tissue, and other tissues. Little is known about osteogenic potential of adipose-derived culture expanded, adherent cells (A-CEAC. This study compares in vivo osteogenic capacity between A-CEAC and bone marrow derived culture expanded, adherent cells (BM-CEAC. Method. A-CEAC and BM-CEAC were isolated from five female sheep and seeded on hydroxyapatite granules prior to subcutaneous implantation in immunodeficient mice. The doses of cells in the implants were 0.5 × 106, 1.0 × 106, or 1.5 × 106 A-CEAC and 0.5 × 106 BM-CEAC, respectively. After eight weeks, bone volume versus total tissue volume (BV/TV was quantified using histomorphometry. Origin of new bone was assessed using human vimentin (HVIM antibody staining. Results. BM-CEAC yielded significantly higher BV/TV than any A-CEAC group, and differences between A-CEAC groups were not statistically significant. HVIM antibody stain was successfully used to identify sheep cells in this model. Conclusion. A-CEAC and BM-CEAC were capable of forming bone, and BM-CEAC yielded significantly higher BV/TV than any A-CEAC group. In vitro treatment to enhance osteogenic capacity of A-CEAC is suggested for further research in ovine bone tissue engineering.

Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells in irradiated bone marrow cell culture in vitro

International Nuclear Information System (INIS)

Fujitake, Hideki; Okamoto, Yuruko; Okubo, Hiroshi; Miyanomae, Takeshi; Kumagai, Keiko; Mori, K.J.

1981-01-01

Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells after irradiation were studied in the long-term culture of mouse bone marrow cells in vitro. No difference was observed in the survival of the stem cells among cultures in which 0 - 10 7 cells were re-inoculated on the adherent cell colonies in the culture flask. Stem cells showed a significant proliferation within 1 week and the number of the stem cells exceeded the control in 3 weeks after irradiation in the cultures with less than 10 6 re-inoculated cells per flask. In contrast, there was a considerable delay in the onset of stem cell proliferation after irradiation in the culture with 10 7 cells per flask. Based on these results, a possibility that a stimulator of stem cell proliferation, released from irradiated stromal cells, is cancelled by an inhibitory factor produced by irradiated or unirradiated haemopoietic cells is postulated. (author)

Development of Collagen/Demineralized Bone Powder Scaffolds and Periosteum-Derived Cells for Bone Tissue Engineering Application

Directory of Open Access Journals (Sweden)

Wilairat Leeanansaksiri

2013-01-01

Full Text Available The aim of this study was to investigate physical and biological properties of collagen (COL and demineralized bone powder (DBP scaffolds for bone tissue engineering. DBP was prepared and divided into three groups, based on various particle sizes: 75–125 µm, 125–250 µm, and 250–500 µm. DBP was homogeneously mixed with type I collagen and three-dimensional scaffolds were constructed, applying chemical crosslinking and lyophilization. Upon culture with human periosteum-derived cells (PD cells, osteogenic differentiation of PD cells was investigated using alkaline phosphatase (ALP activity and calcium assay kits. The physical properties of the COL/DBP scaffolds were obviously different from COL scaffolds, irrespective of the size of DBP. In addition, PD cells cultured with COL scaffolds showed significantly higher cell adhesion and proliferation than those with COL/DBP scaffolds. In contrast, COL/DBP scaffolds exhibited greater osteoinductive potential than COL scaffolds. The PD cells with COL/DBP scaffolds possessed higher ALP activity than those with COL scaffolds. PD cells cultured with COL/DBP scaffolds with 250–500 mm particle size yielded the maximum calcium deposition. In conclusion, PD cells cultured on the scaffolds could exhibit osteoinductive potential. The composite scaffold of COL/DBP with 250–500 mm particle size could be considered a potential bone tissue engineering implant.

Case of Six-Year Disease-Free Survival with Undifferentiated Carcinoma of the Pancreas

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Hiroyuki Saito

2016-08-01

Full Text Available Undifferentiated carcinoma of the pancreas (UDC is rare and has a dismal prognosis. Here, we report a case of 6-year disease-free survival with a mixed type of UDC and UDC with osteoclast-like giant cells, with a high mitotic index as well as perineural, lymphatic, vessel, and diaphragmatic invasion. The patient underwent radical distal pancreatectomy and was subsequently treated with adjuvant chemotherapy using gemcitabine plus S-1 followed by maintenance chemotherapy with oral tegafur-uracil. The patient has been doing well with no evidence of recurrence for more than 6 years after surgery.

Heterogeneity of the cytokinome in undifferentiated arthritis progressing to rheumatoid arthritis and its change in the course of therapy. Move toward personalized medicine.

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Brzustewicz, Edyta; Bzoma, Izabella; Daca, Agnieszka; Szarecka, Maria; Bykowska, Malgorzata Sochocka; Witkowski, Jacek M; Bryl, Ewa

2017-09-01

To conduct a comprehensive analysis of cytokine concentrations in sera and mononuclear cell supernatants in order to examine inter- and intra-individual cytokine variations in undifferentiated arthritis progressing to rheumatoid arthritis and healthy control groups. Patients with UA (undifferentiated arthritis) developing RA (rheumatoid arthritis) (UA→RA) (n=16) and healthy controls (n=16) were enrolled into the study. UA→RA patients were followed up for six months since the final RA diagnosis. Cytokines IFN-γ, IL-10, TNF, IL-17A, IL-6, IL-1β, IL-2 in sera and mononuclear cell supernatants in 72h and 120h culture variants with- and without anti-CD3 stimulations were assayed using flow cytometric bead array. The cytokine profile of UA→RA differs from the healthy individual cytokine profile. It is possible to observe specific cytokine pattern characterizing each patient, which alters during course of disease. Specifically, we can distinguish three UA→RA cohorts: the group of patients susceptible to the therapy, characterized by the drop of cytokine levels between 1st and 3rd visit with visible decrease of cytokines in 2nd visit and then secondary slighter increase in 3rd visit; the group of patients refractory or clinically worsening on the therapy, characterized by the highest cytokine levels at 2nd visit with secondary decrease in 3rd visit; and the group of patients with variable responses to the therapy without any specific common cytokine pattern. The cytokine patterns in supernatants of PBMC stimulated anti-CD3 for 72h and 120h are very similar. The personal profile including multiplexed cytokine patterns in serum and supernatant may be potentially used for optimization of therapy introduction and monitoring. Copyright © 2017 Elsevier Ltd. All rights reserved.

Marginal zinc deficiency in pregnant rats impairs bone matrix formation and bone mineralization in their neonates.

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Nagata, Masashi; Kayanoma, Megumu; Takahashi, Takeshi; Kaneko, Tetsuo; Hara, Hiroshi

2011-08-01

Zinc (Zn) deficiency during pregnancy may result in a variety of defects in the offspring. We evaluated the influence of marginal Zn deficiency during pregnancy on neonatal bone status. Nine-week-old male Sprague-Dawley rats were divided into two groups and fed AIN-93G-based experimental diets containing 35 mg Zn/kg (Zn adequately supplied, N) or 7 mg Zn/kg (low level of Zn, L) from 14-day preconception to 20 days of gestation, that is, 1 day before normal delivery. Neonates were delivered by cesarean section. Litter size and neonate weight were not different between the two groups. However, in the L-diet-fed dam group, bone matrix formation in isolated neonatal calvaria culture was clearly impaired and was not recovered by the addition of Zn into the culture media. Additionally, serum concentration of osteocalcin, as a bone formation parameter, was lower in neonates from the L-diet-fed dam group. Impaired bone mineralization was observed with a significantly lower content of phosphorus in neonate femurs from L-diet-fed dams compared with those from N-diet-fed dams. Moreover, Zn content in the femur and calvaria of neonates from the L-diet group was lower than that of the N-diet-fed group. In the marginally Zn-deficient dams, femoral Zn content, serum concentrations of Zn, and osteocalcin were reduced when compared with control dams. We conclude that maternal Zn deficiency causes impairment of bone matrix formation and bone mineralization in neonates, implying the importance of Zn intake during pregnancy for proper bone development of offspring.

Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM.

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Deegan, Anthony J; Cinque, Gianfelice; Wehbe, Katia; Konduru, Sandeep; Yang, Ying

2015-02-01

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process

Risk factors for the occurrence of undifferentiated carcinoma of nasopharyngeal type: A case-control study

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Nešić Vladimir; Šipetić Sandra; Vlajinac Hristina; Stošić-Divjak Svetlana; Ješić Snežana

2010-01-01

Introduction. The incidence rate of nasopharyngeal carcinoma in Serbia is less than one per 100,000 citizens, which classifies it as a region with low incidence for this disease. Objective. The aim of this study was to test some hypotheses of the risk factors for undifferentiated carcinoma of nasopharyngeal type (UCNT) in the low incidence population. Methods. A case-control study was used for the research. The study included 45 cases with histopathological diagnosis of UCNT and 90 controls. ...

Effectiveness of mesenchymal stems cells cultured by hanging drop vs. conventional culturing on the repair of hypoxic-ischemic-damaged mouse brains, measured by stemness gene expression

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Lou Yongli

2016-01-01

Full Text Available In this study, we investigated the therapeutic effects of Human Mesenchymal Stem Cells (hMSCs cultured by hanging drop and conventional culturing methods on cerebellar repair in hypoxic-ischemic (HI brain injured mice. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR was used to analyze the expression levels of three stemness genes, Oct4, Sox2 and Nanog, and the migration related gene CXCR4. MSC prepared by hanging drop or conventional techniques were administered intranasally to nine day old mice, and analyzed by MRI at day 28. Results indicate that the MSCs, especially the hanging drop cultured MSCs, significantly improved the mice’s cerebellar damage repair. MSCs derived from the hanging drop culture were smaller than those from the conventional culture. The gene expression levels were significantly increased for the MSCs derived from the hanging drop culture. The mechanism might relate to the fact that the hanging drop cultured MSCs can be kept in an undifferentiated state, resulting in its higher expression level of migration receptor of CXCR4.

Towards modular bone tissue engineering using Ti-Co-doped phosphate glass microspheres: cytocompatibility and dynamic culture studies.

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Peticone, Carlotta; De Silva Thompson, David; Owens, Gareth J; Kim, Hae-Won; Micheletti, Martina; Knowles, Jonathan C; Wall, Ivan

2017-09-01

The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti-Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue

Atlantoaxial subluxation as an early manifestation in an adolescent with undifferentiated spondyloarthritis: a case report and review of the literature

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Jea Andrew

2011-07-01

Full Text Available Abstract Introduction Atlantoaxial instability has been described as a manifestation of ankylosing spondylitis (juvenile and adult onset, reactive arthritis, juvenile idiopathic arthritis, and rheumatoid arthritis; however, it has rarely been reported as an early manifestation of these disorders. We present this case report to increase awareness of the condition in the hope that earlier recognition of this disease may prevent further serious injury. Case presentation We report the case of a 17-year-old Hispanic adolescent woman who was initially diagnosed with undifferentiated spondyloarthritis due to peripheral arthritis, enthesitis, a positive human leukocyte antigen B27 result, and inflammatory spinal pain lasting two months. Our patient experienced persistent and worsening occipitocervical pain and signs of myelopathy three months after diagnosis; consequently, we found atlantoaxial instability along with cervical spine bone erosion and pannus formation. She was treated surgically with a C1-2 posterior instrumented fusion and at six weeks post-operatively was started on tumor necrosis factor α blockade. Her occipitocervical symptoms subsided following surgery and initiation of immunomodulation. Conclusions Our report serves to emphasize to pediatric and adult general practitioners, pediatricians, internists, family physicians, pediatric and adult rheumatologists and spine surgeons that atlantoaxial subluxation may be an early manifestation of spondyloarthritis, and that the condition is treatable by surgical intervention and immunomodulation.

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

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2012-01-01

Introduction The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs. Methods Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR. Results Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of

PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

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A.M. NOSOV

2014-06-01

Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures. 

CT findings of primary undifferentiated pleomorphic sarcoma in the small bowel: A case report

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Kim, Youe Ree; Lee, Young Hwan; Yoon, Kwon Ha; Yun, Ki Jung [Wonkwang University School of Medicine and Hospital, Institute of Wonkwang Medical Science, Iksan (Korea, Republic of)

2015-11-15

Undifferentiated pleomorphic sarcoma (UPS), previously known as malignant fibrous histiocytoma, is a soft tissue sarcoma arising from mesenchymal tissue of the body. UPS of the gastrointestinal tract is known to be rare and only a few cases have been reported in the literature. Based on our case and review of the other relevant literature, the CT findings of primary UPS of the small bowel included nodular bowel wall thickening with homogeneous enhancement. It presents as a rapidly growing tumor without bowel obstruction, and it may be accompanied by distant metastasis.

Comparison of Osteogenesis between Adipose-Derived Mesenchymal Stem Cells and Their Sheets on Poly-ε-Caprolactone/β-Tricalcium Phosphate Composite Scaffolds in Canine Bone Defects

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Yongsun Kim

2016-01-01

Full Text Available Multipotent mesenchymal stem cells (MSCs and MSC sheets have effective potentials of bone regeneration. Composite polymer/ceramic scaffolds such as poly-ε-caprolactone (PCL/β-tricalcium phosphate (β-TCP are widely used to repair large bone defects. The present study investigated the in vitro osteogenic potential of canine adipose-derived MSCs (Ad-MSCs and Ad-MSC sheets. Composite PCL/β-TCP scaffolds seeded with Ad-MSCs or wrapped with osteogenic Ad-MSC sheets (OCS were also fabricated and their osteogenic potential was assessed following transplantation into critical-sized bone defects in dogs. The alkaline phosphatase (ALP activity of osteogenic Ad-MSCs (O-MSCs and OCS was significantly higher than that of undifferentiated Ad-MSCs (U-MSCs. The ALP, runt-related transcription factor 2, osteopontin, and bone morphogenetic protein 7 mRNA levels were upregulated in O-MSCs and OCS as compared to U-MSCs. In a segmental bone defect, the amount of newly formed bone was greater in PCL/β-TCP/OCS and PCL/β-TCP/O-MSCs/OCS than in the other groups. The OCS exhibit strong osteogenic capacity, and OCS combined with a PCL/β-TCP composite scaffold stimulated new bone formation in a critical-sized bone defect. These results suggest that the PCL/β-TCP/OCS composite has potential clinical applications in bone regeneration and can be used as an alternative treatment modality in bone tissue engineering.

Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

International Nuclear Information System (INIS)

Ransjoe, M.; Lerner, U.H.

1990-01-01

Calcitonin is a well known inhibitor of osteoclastic bone resortion, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called ''escape from inhibition'' phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45 Ca. Two proteon kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45 Ca release in 120-h cultures at a concentration of 10 nmul/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any ''escape from inhibition''. This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E 2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption. (author)

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate Bone Cement on Mechanical Properties and Bioactivity.

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Tao Li

Full Text Available This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA bone cement after addition of the nano-hydroxyapatite(HA coated bone collagen (mineralized collagen, MC.The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis.15.0%(wt impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA.MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values.

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

International Nuclear Information System (INIS)

Otsuru, Satoru; Tamai, Katsuto; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

2007-01-01

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

A case report of prostate cancer metastasis to the stomach resembling undifferentiated-type early gastric cancer.

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Inagaki, Chiaki; Suzuki, Takuto; Kitagawa, Yoshiyasu; Hara, Taro; Yamaguchi, Taketo

2017-08-07

Occurrence of metastatic cancer to the stomach is rare, particularly in patients with prostate cancer. Gastric metastasis generally presents as a solitary and submucosal lesion with a central depression. We describe a case of gastric metastasis arising from prostate cancer, which is almost indistinguishable from the undifferentiated-type gastric cancer. A definitive diagnosis was not made until endoscopic resection. On performing both conventional and magnifying endoscopies, the lesion appeared to be slightly depressed and discolored area and it could not be distinguished from undifferentiated early gastric cancer. Biopsy from the lesion was negative for immunohistochemical staining of prostate-specific antigen, a sensitive and specific marker for prostate cancer. Thus, false initial diagnosis of an early primary gastric cancer was made and endoscopic submucosal dissection was performed. Pathological findings from the resected specimen aroused suspicion of a metastatic lesion. Consequently, immunostaining was performed. The lesion was positive for prostate-specific acid phosphatase and negative for prostate-specific antigen, cytokeratin 7, and cytokeratin 20. Accordingly, the final diagnosis was a metastatic gastric lesion originating from prostate cancer. In this patient, the definitive diagnosis as a metastatic lesion was difficult due to its unusual endoscopic appearance and the negative stain for prostate-specific antigen. We postulate that both of these are consequences of hormonal therapy against prostate cancer.

Affect of Bioglass {sup trademark} repeat dosage on mineralisation of embryonic bone 'in vitro'

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Maroothynaden, J. [Imperial Coll. of Medicine, London (United Kingdom). Microgravity Tissue Engineering Lab.; Hench, L.L. [Imperial Coll. of Science, Technology and Medicine, London (United Kingdom). Dept. of Materials

2001-07-01

Utilising 45S5 Bioglass {sup trademark} extracts, as described previously, 16-day gestation embryonic mouse long-bones were cultured for 4-days while exposed to the same Bioglass{sup circledR} soluble extract solution for two different exposure times. In the first culture, all embryonic femurs were exposed to fresh 45S5 Bioglass {sup trademark} extract every 98 hours. In the second, the long-bones were exposed to fresh 45S5 Bioglass {sup trademark} extract solution every 48 hours. A simultaneous control culture was performed. All embryonic long-bone cultures mineralised after 4-days culture. Increasing the frequency of 45S5 Bioglass {sup trademark} exposure, from one exposure every 96 hrs to fresh exposures every 48 hrs, significantly increased the length and mineral content of the embryonic long-bones. (orig.)

Osteoclast TGF-β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

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Weivoda, Megan M; Ruan, Ming; Pederson, Larry; Hachfeld, Christine; Davey, Rachel A; Zajac, Jeffrey D; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

2016-01-01

Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclastspecific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. PMID:26108893

Mechanisms Inducing Low Bone Density in Duchenne Muscular Dystrophy in Mice and Humans

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Rufo, Anna; Del Fattore, Andrea; Capulli, Mattia; Carvello, Francesco; De Pasquale, Loredana; Ferrari, Serge; Pierroz, Dominique; Morandi, Lucia; De Simone, Michele; Rucci, Nadia; Bertini, Enrico; Bianchi, Maria Luisa; De Benedetti, Fabrizio; Teti, Anna

2011-01-01

Patients affected by Duchenne muscular dystrophy (DMD) and dystrophic MDX mice were investigated in this study for their bone phenotype and systemic regulators of bone turnover. Micro–computed tomographic (µCT) and histomorphometric analyses showed reduced bone mass and higher osteoclast and bone resorption parameters in MDX mice compared with wild-type mice, whereas osteoblast parameters and mineral apposition rate were lower. In a panel of circulating pro-osteoclastogenic cytokines evaluated in the MDX sera, interleukin 6 (IL-6) was increased compared with wild-type mice. Likewise, DMD patients showed low bone mineral density (BMD) Z-scores and high bone-resorption marker and serum IL-6. Human primary osteoblasts from healthy donors incubated with 10% sera from DMD patients showed decreased nodule mineralization. Many osteogenic genes were downregulated in these cultures, including osterix and osteocalcin, by a mechanism blunted by an IL-6-neutralizing antibody. In contrast, the mRNAs of osteoclastogenic cytokines IL6, IL11, inhibin-βA, and TGFβ2 were increased, although only IL-6 was found to be high in the circulation. Consistently, enhancement of osteoclastogenesis was noted in cultures of circulating mononuclear precursors from DMD patients or from healthy donors cultured in the presence of DMD sera or IL-6. Circulating IL-6 also played a dominant role in osteoclast formation because ex vivo wild-type calvarial bones cultured with 10% sera of MDX mice showed increase osteoclast and bone-resorption parameters that were dampen by treatment with an IL-6 antibody. These results point to IL-6 as an important mediator of bone loss in DMD and suggest that targeted anti-IL-6 therapy may have a positive impact on the bone phenotype in these patients. © 2011 American Society for Bone and Mineral Research PMID:21509823

Numerical simulation of fluid field and in vitro three-dimensional fabrication of tissue-engineered bones in a rotating bioreactor and in vivo implantation for repairing segmental bone defects.

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Song, Kedong; Wang, Hai; Zhang, Bowen; Lim, Mayasari; Liu, Yingchao; Liu, Tianqing

2013-03-01

In this paper, two-dimensional flow field simulation was conducted to determine shear stresses and velocity profiles for bone tissue engineering in a rotating wall vessel bioreactor (RWVB). In addition, in vitro three-dimensional fabrication of tissue-engineered bones was carried out in optimized bioreactor conditions, and in vivo implantation using fabricated bones was performed for segmental bone defects of Zelanian rabbits. The distribution of dynamic pressure, total pressure, shear stress, and velocity within the culture chamber was calculated for different scaffold locations. According to the simulation results, the dynamic pressure, velocity, and shear stress around the surface of cell-scaffold construction periodically changed at different locations of the RWVB, which could result in periodical stress stimulation for fabricated tissue constructs. However, overall shear stresses were relatively low, and the fluid velocities were uniform in the bioreactor. Our in vitro experiments showed that the number of cells cultured in the RWVB was five times higher than those cultured in a T-flask. The tissue-engineered bones grew very well in the RWVB. This study demonstrates that stress stimulation in an RWVB can be beneficial for cell/bio-derived bone constructs fabricated in an RWVB, with an application for repairing segmental bone defects.

Biochemical markers of embryogenesis in tissue cultures of the maize inbred B73

International Nuclear Information System (INIS)

Everett, N.P.; Wach, M.J.; Ashworth, D.J.

1985-01-01

Stable embryogenic, organogenic and undifferentiated cell lines of the maize (Zea mays L.) inbred B73 were used to assess the value of using isozyme analyses and the composition of secreted polysaccharides to identify embryogenic cells. Esterase, glutamate dehydrogenase, alcohol dehydrogenase and β-glucosidase all possessed developmentally regulated isozymes but only esterase and glutamate dehydrogenase could be used to distinguish between embryogenic and shoot-forming cultures. Embryogenic callus and suspension cultures secreted a mucilagenous polysaccharide whose production was stimulated by 2, 4-dichlorophenozyacetic acid (2, 4-D). The polysaccharide was different from root slime and corn hull gum and may be related to the 'cementing layer' in maize kernels (author)

Assessment of Effects of Si-Ca-P Biphasic Ceramic on the Osteogenic Differentiation of a Population of Multipotent Adult Human Stem Cells

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Patricia Ros-Tárraga

2016-11-01

Full Text Available A new type of bioceramic with osteogenic properties, suitable for hard tissue regeneration, was synthesised. The ceramic was designed and obtained in the Nurse’s A-phase-silicocarnotite subsystem. The selected composition was that corresponding to the eutectoid 28.39 wt % Nurse’s A-phase-71.61 wt % silicocarnotite invariant point. We report the effect of Nurse’s A-phase-silicocarnotite ceramic on the capacity of multipotent adult human mesenchymal stem cells (ahMSCs cultured under experimental conditions, known to adhere, proliferate and differentiate into osteoblast lineage cells. The results at long-term culture (28 days on the material confirmed that the undifferentiated ahMSCs cultured and in contact with the material surface adhered, spread, proliferated, and produced a mineralised extracellular matrix on the studied ceramic, and finally acquired an osteoblastic phenotype. These findings indicate that it underwent an osteoblast differentiation process. All these findings were more significant than when cells were grown on plastic, in the presence and absence of this osteogenic supplement, and were more evident when this supplement was present in the growth medium (GM. The ceramic evaluated herein was bioactive, cytocompatible and capable of promoting the proliferation and differentiation of undifferentiated ahMSCs into osteoblasts, which may be important for bone integration into the clinical setting.

Nano-ceramic composite scaffolds for bioreactor-based bone engineering.

Science.gov (United States)

Lv, Qing; Deng, Meng; Ulery, Bret D; Nair, Lakshmi S; Laurencin, Cato T

2013-08-01

Composites of biodegradable polymers and bioactive ceramics are candidates for tissue-engineered scaffolds that closely match the properties of bone. We previously developed a porous, three-dimensional poly (D,L-lactide-co-glycolide) (PLAGA)/nanohydroxyapatite (n-HA) scaffold as a potential bone tissue engineering matrix suitable for high-aspect ratio vessel (HARV) bioreactor applications. However, the physical and cellular properties of this scaffold are unknown. The present study aims to evaluate the effect of n-HA in modulating PLAGA scaffold properties and human mesenchymal stem cell (HMSC) responses in a HARV bioreactor. By comparing PLAGA/n-HA and PLAGA scaffolds, we asked whether incorporation of n-HA (1) accelerates scaffold degradation and compromises mechanical integrity; (2) promotes HMSC proliferation and differentiation; and (3) enhances HMSC mineralization when cultured in HARV bioreactors. PLAGA/n-HA scaffolds (total number = 48) were loaded into HARV bioreactors for 6 weeks and monitored for mass, molecular weight, mechanical, and morphological changes. HMSCs were seeded on PLAGA/n-HA scaffolds (total number = 38) and cultured in HARV bioreactors for 28 days. Cell migration, proliferation, osteogenic differentiation, and mineralization were characterized at four selected time points. The same amount of PLAGA scaffolds were used as controls. The incorporation of n-HA did not alter the scaffold degradation pattern. PLAGA/n-HA scaffolds maintained their mechanical integrity throughout the 6 weeks in the dynamic culture environment. HMSCs seeded on PLAGA/n-HA scaffolds showed elevated proliferation, expression of osteogenic phenotypic markers, and mineral deposition as compared with cells seeded on PLAGA scaffolds. HMSCs migrated into the scaffold center with nearly uniform cell and extracellular matrix distribution in the scaffold interior. The combination of PLAGA/n-HA scaffolds with HMSCs in HARV bioreactors may allow for the generation of engineered

Comparison of potentials between stem cells isolated from human anterior cruciate ligament and bone marrow for ligament tissue engineering.

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Cheng, Ming-Te; Liu, Chien-Lin; Chen, Tain-Hsiung; Lee, Oscar K

2010-07-01

We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

Immortalized porcine mesenchymal cells derived from nasal mucosa, lungs, lymph nodes, spleen and bone marrow retain their stemness properties and trigger the expression of siglec-1 in co-cultured blood monocytic cells.

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Garba, Abubakar; Desmarets, Lowiese M B; Acar, Delphine D; Devriendt, Bert; Nauwynck, Hans J

2017-01-01

Mesenchymal stromal cells have been isolated from different sources. They are multipotent cells capable of differentiating into many different cell types, including osteocytes, chondrocytes and adipocytes. They possess a therapeutic potential in the management of immune disorders and the repair of damaged tissues. Previous work in our laboratory showed an increase of the percentages of CD172a+, CD14+, CD163+, Siglec-1+, CD4+ and CD8+ hematopoietic cells, when co-cultured with immortalized mesenchymal cells derived from bone marrow. The present work aimed to demonstrate the stemness properties of SV40-immortalized mesenchymal cells derived from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow and their immunomodulatory effect on blood monocytes. Mesenchymal cells from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow were isolated and successfully immortalized using simian virus 40 large T antigen (SV40LT) and later, co-cultured with blood monocytes, in order to examine their differentiation stage (expression of Siglec-1). Flow cytometric analysis revealed that the five mesenchymal cell lines were positive for mesenchymal cell markers CD105, CD44, CD90 and CD29, but lacked the expression of myeloid cell markers CD16 and CD11b. Growth analysis of the cells demonstrated that bone marrow derived-mesenchymal cells proliferated faster compared with those derived from the other tissues. All five mesenchymal cell lines co-cultured with blood monocytes for 1, 2 and 7 days triggered the expression of siglec-1 in the monocytes. In contrast, no siglec-1+ cells were observed in monocyte cultures without mesenchymal cell lines. Mesenchymal cells isolated from nasal mucosa, lungs, spleen, lymph nodes and bone marrow were successfully immortalized and these cell lines retained their stemness properties and displayed immunomodulatory effects on blood monocytes.

Design and validation of a dynamic cell-culture system for bone biology research and exogenous tissue-engineering applications.

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Allori, Alexander C; Davidson, Edward H; Reformat, Derek D; Sailon, Alexander M; Freeman, James; Vaughan, Adam; Wootton, David; Clark, Elizabeth; Ricci, John L; Warren, Stephen M

2016-10-01

Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell-culture methods are ill-suited to study the intricacies of bone biology because they ignore the three-dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2-3 mm thickness. In this project, a flow-perfusion system was custom-designed to overcome these limitations: eight adaptable chambers housed cylindrical cell-seeded scaffolds measuring 12 or 24 mm in diameter and 1-10 mm in thickness. The porous scaffolds were manufactured using a three-dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi-channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas-permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor-scaffold system, using 12 × 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Using bones to shape stones: MIS 9 bone retouchers at both edges of the Mediterranean Sea.

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Ruth Blasco

Full Text Available A significant challenge in Prehistory is to understand the mechanisms involved in the behavioural evolution of human groups. The degree of technological and cultural development of prehistoric groups is assessed mainly through stone tools. However, other elements can provide valuable information as well. This paper presents two bone retouchers dated to the Middle Pleistocene MIS 9 used for the shaping of lithic artefacts. Originating from Bolomor Cave (Spain and Qesem Cave (Israel, these two bone retouchers are among the earliest of the Old World. Although the emergence of such tools might be found in the latest phases of the Acheulean, their widespread use seems to coincide with independently emergent post-Acheulean cultural complexes at both ends of the Mediterranean Sea: the post-Acheulean/pre-Mousterian of Western Europe and the Acheulo Yabrudian Cultural Complex of the Levant. Both entities seem to reflect convergent processes that may be viewed in a wider cultural context as reflecting new technology-related behavioural patterns as well as new perceptions in stone tool manufacturing.

[Linkage analysis of susceptibility loci in 2 target chromosomes in pedigrees with paranoid schizophrenia and undifferentiated schizophrenia].

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Zeng, Li-ping; Hu, Zheng-mao; Mu, Li-li; Mei, Gui-sen; Lu, Xiu-ling; Zheng, Yong-jun; Li, Pei-jian; Zhang, Ying-xue; Pan, Qian; Long, Zhi-gao; Dai, He-ping; Zhang, Zhuo-hua; Xia, Jia-hui; Zhao, Jing-ping; Xia, Kun

2011-06-01

To investigate the relationship of susceptibility loci in chromosomes 1q21-25 and 6p21-25 and schizophrenia subtypes in Chinese population. A genomic scan and parametric and non-parametric analyses were performed on 242 individuals from 36 schizophrenia pedigrees, including 19 paranoid schizophrenia and 17 undifferentiated schizophrenia pedigrees, from Henan province of China using 5 microsatellite markers in the chromosome region 1q21-25 and 8 microsatellite markers in the chromosome region 6p21-25, which were the candidates of previous studies. All affected subjects were diagnosed and typed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR; American Psychiatric Association, 2000). All subjects signed informed consent. In chromosome 1, parametric analysis under the dominant inheritance mode of all 36 pedigrees showed that the maximum multi-point heterogeneity Log of odds score method (HLOD) score was 1.33 (α = 0.38). The non-parametric analysis and the single point and multi-point nonparametric linkage (NPL) scores suggested linkage at D1S484, D1S2878, and D1S196. In the 19 paranoid schizophrenias pedigrees, linkage was not observed for any of the 5 markers. In the 17 undifferentiated schizophrenia pedigrees, the multi-point NPL score was 1.60 (P= 0.0367) at D1S484. The single point NPL score was 1.95(P= 0.0145) and the multi-point NPL score was 2.39 (P= 0.0041) at D1S2878. Additionally, the multi-point NPL score was 1.74 (P= 0.0255) at D1S196. These same three loci showed suggestive linkage during the integrative analysis of all 36 pedigrees. In chromosome 6, parametric linkage analysis under the dominant and recessive inheritance and the non-parametric linkage analysis of all 36 pedigrees and the 17 undifferentiated schizophrenia pedigrees, linkage was not observed for any of the 8 markers. In the 19 paranoid schizophrenias pedigrees, parametric analysis showed that under recessive

Rhus javanica Gall Extract Inhibits the Differentiation of Bone Marrow-Derived Osteoclasts and Ovariectomy-Induced Bone Loss

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Tae-Ho Kim

2016-01-01

Full Text Available Inhibition of osteoclast differentiation and bone resorption is a therapeutic strategy for the management of postmenopausal bone loss. This study investigated the effects of Rhus javanica (R. javanica extracts on bone marrow cultures to develop agents from natural sources that may prevent osteoclastogenesis. Extracts of R. javanica (eGr cocoons spun by Rhus javanica (Bell. Baker inhibited the osteoclast differentiation and bone resorption. The effects of aqueous extract (aeGr or 100% ethanolic extract (eeGr on ovariectomy- (OVX- induced bone loss were investigated by various biochemical assays. Furthermore, microcomputed tomography (µCT was performed to study bone remodeling. Oral administration of eGr (30 mg or 100 mg/kg/day for 6 weeks augmented the inhibition of femoral bone mineral density (BMD, bone mineral content (BMC, and other factors involved in bone remodeling when compared to OVX controls. Additionally, eGr slightly decreased bone turnover markers that were increased by OVX. Therefore, it may be suggested that the protective effects of eGr could have originated from the suppression of OVX-induced increase in bone turnover. Collectively, the findings of this study indicate that eGr has potential to activate bone remodeling by inhibiting osteoclast differentiation and bone loss.

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

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Jensen Jonas

2016-01-01

Full Text Available Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs was compared with that of dental pulp-derived stromal cells (DPSCs in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D polycaprolactone (PCL – hyaluronic acid – tricalcium phosphate (HT-PCL scaffold. Population doubling (PD, alkaline phosphatase (ALP activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1 empty defects vs. HT-PCL scaffolds; (2 PCL scaffolds vs. HT-PCL scaffolds; and (3 autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV were assessed with micro-computed tomography (μCT and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations

International Nuclear Information System (INIS)

Taga, Masataka; Shiraishi, Kazunori; Shimura, Tsutomu; Uematsu, Norio; Kato, Tomohisa; Niwa, Ohtsura; Nishimune, Yoshitake; Aizawa, Shinichi; Oshimura, Mitsuo

2000-01-01

The effect of caffeine was studied on the radioresponses of undifferentiated mouse embryonal carcinoma cells (EC cells) with or without the functional p53. The radioresponses studied included radiosensitivity, the activation of p53, apoptosis with characteristic DNA ladder formation and cell cycle progression. An undifferentiated mouse EC cell line, ECA2, and a newly established p53-deficient EC cell line, p53δ, were used in the present study. The status of the p53 gene did not significantly affect the colony survivals of undifferentiated EC cells to X-rays and UV. Although a post-irradiation treatment with caffeine sensitized both lines to X-rays marginally, the sensitization was prominent for UV regardless of the p53 status of the cells. The activation of a p53 responsible lacZ reporter construct was observed in stably transfected ECA2 cells after X-ray and UV irradiations. Caffeine suppressed the X-ray induced activation of the lacZ reporter, while it drastically enhanced the activation after UV irradiation. X-rays and UV readily triggered the apoptosis of ECA2 cells with the characteristic DNA ladder. Although UV-induced DNA ladder formation was enhanced by caffeine, that induced by X-rays was unaffected. Therefore, the effects of caffeine on the p53-dependent radioresponses were found to be agent specific: suppression for the X-ray induced and augmentation for the UV induced. In contrast to p53-proficient ECA2 cells, smear-like DNA degradation was observed for irradiated p53δ cells, suggesting the presence of a mode of cell death without DNA ladder formation. UV induction of the smear-like DNA degradation was enhanced in the presence of caffeine. Regardless of the state of the p53 gene, G1/S arrest was not observed in X-ray and UV irradiated EC cells. X-rays induced G2/M arrest in both lines, which was abrogated by caffeine, while G2/M arrest after UV was unaffected by a caffeine treatment. These results indicate that the radioresponses of undifferentiated

The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations

Energy Technology Data Exchange (ETDEWEB)

Taga, Masataka; Shiraishi, Kazunori; Shimura, Tsutomu; Uematsu, Norio; Kato, Tomohisa; Niwa, Ohtsura [Kyoto Univ. (Japan). Radiation Biology Center; Nishimune, Yoshitake; Aizawa, Shinichi; Oshimura, Mitsuo

2000-09-01

The effect of caffeine was studied on the radioresponses of undifferentiated mouse embryonal carcinoma cells (EC cells) with or without the functional p53. The radioresponses studied included radiosensitivity, the activation of p53, apoptosis with characteristic DNA ladder formation and cell cycle progression. An undifferentiated mouse EC cell line, ECA2, and a newly established p53-deficient EC cell line, p53{delta}, were used in the present study. The status of the p53 gene did not significantly affect the colony survivals of undifferentiated EC cells to X-rays and UV. Although a post-irradiation treatment with caffeine sensitized both lines to X-rays marginally, the sensitization was prominent for UV regardless of the p53 status of the cells. The activation of a p53 responsible lacZ reporter construct was observed in stably transfected ECA2 cells after X-ray and UV irradiations. Caffeine suppressed the X-ray induced activation of the lacZ reporter, while it drastically enhanced the activation after UV irradiation. X-rays and UV readily triggered the apoptosis of ECA2 cells with the characteristic DNA ladder. Although UV-induced DNA ladder formation was enhanced by caffeine, that induced by X-rays was unaffected. Therefore, the effects of caffeine on the p53-dependent radioresponses were found to be agent specific: suppression for the X-ray induced and augmentation for the UV induced. In contrast to p53-proficient ECA2 cells, smear-like DNA degradation was observed for irradiated p53{delta} cells, suggesting the presence of a mode of cell death without DNA ladder formation. UV induction of the smear-like DNA degradation was enhanced in the presence of caffeine. Regardless of the state of the p53 gene, G1/S arrest was not observed in X-ray and UV irradiated EC cells. X-rays induced G2/M arrest in both lines, which was abrogated by caffeine, while G2/M arrest after UV was unaffected by a caffeine treatment. These results indicate that the radioresponses of

Acute erythroblastic leukemia presenting as acute undifferentiated leukemia: a report of two cases with ultrastructural features.

Science.gov (United States)

Reiffers, J; Bernard, P; Larrue, J; Dachary, D; David, B; Boisseau, M; Broustet, A

1985-01-01

This report describes two elderly patients with acute leukemia in which blast cells were undifferentiated with conventional light microscopy (L.M.) and cytochemistry. Blast cells were identified as belonging to the erythroblastic line by their ultrastructural features: glycogen deposits, lipidic vacuoles, cytoplasmic ferritin molecules and rhopheocytotic invagination. Moreover, blast cells were surrounding a central macrophage. Thus, these two patients had acute erythroblastic leukemia which differs from erythroleukemia (M6 of FAB classification) in which blast cells present myeloblastic characteristics.

A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells.

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Watts, Ashlee E; Ackerman-Yost, Jeremy C; Nixon, Alan J

2013-10-01

To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X). alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5×10(5) cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels. equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems.

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

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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.

Multicenter validation of the value of BASFI and BASDAI in Chinese ankylosing spondylitis and undifferentiated spondyloarthropathy patients

OpenAIRE

Lin, Zhiming; Gu, Jieruo; He, Peigen; Gao, Jiesheng; Zuo, Xiaoxia; Ye, Zhizhong; Shao, Fengmin; Zhan, Feng; Lin, Jinying; Li, Li; Wei, Yanlin; Xu, Manlong; Liao, Zetao; Lin, Qu

2009-01-01

The objectives of this study were to evaluate the reliability of Bath ankylosing spondylitis functional index (BASFI) and Bath ankylosing spondylitis disease activity index (BASDAI) in Chinese ankylosing spondylitis (AS) and undifferentiated spondyloarthropathy (USpA) patients. 664 AS patients by the revised New York criteria for AS and 252 USpA patients by the European Spondyloarthropathy Study Group criteria were enrolled. BASDAI and BASFI questionnaires were translated into Chinese. Partic...

Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation.

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Menon, Alessandra; Creo, Pasquale; Piccoli, Marco; Bergante, Sonia; Conforti, Erika; Banfi, Giuseppe; Randelli, Pietro; Anastasia, Luigi

2018-01-01

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the "hypoxic niches" present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.

Chemical Activation of the Hypoxia-Inducible Factor Reversibly Reduces Tendon Stem Cell Proliferation, Inhibits Their Differentiation, and Maintains Cell Undifferentiation

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Alessandra Menon

2018-01-01

Full Text Available Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21% has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF, the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the “hypoxic niches” present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.

Cloning higher plants from aseptically cultured tissues and cells

Science.gov (United States)

Krikorian, A. D.

1982-01-01

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

Novel Therapy for Bone Regeneration in Large Segmental Defects

Science.gov (United States)

2017-12-01

can maintain bone length and allow successfully regeneration in segmental defects. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bone...pre- formed endothelial networks, as the MSCs can act as pericytes to the newly formed blood vessels. Pirraco et al. [159] also cultured ECs and...AWARD NUMBER: W81XWH-13-1-0407 TITLE: Novel Therapy for Bone Regeneration in Large Segmental Defects PRINCIPAL INVESTIGATOR: Melissa Kacena

[Undifferentiated cutaneous angiosarcoma of the head: identification by the endothelial marker Ulex europaeus agglutinin I].

Science.gov (United States)

Bork, K; Fries, J; Hoede, N; Korting, G W; Dienes, P

1985-06-01

Cutaneous angiosarcoma of the head is a rare tumor of the elderly and can occur in an undifferentiated form without any clinical or histological signs of the vascular origin of this tumor. In these cases, the tumor can be identified by using endothelial cell markers, such as factor-VIII-related antigen and ulex europaeus agglutinin I, in an immunofluorescence technique or a peroxidase-antiperoxidase method. A 78-year-old patient is described who died within 18 months from such a tumor, which was diagnosed using the endothelial cell marker, ulex europaeus agglutinin I.

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

OpenAIRE

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomita...

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration

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Rubén Aquino-Martínez

2017-11-01

Full Text Available Abstract Background Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4 on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved. Methods A circular calvarial bone defect (5 mm diameter was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR. Results CaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration. Conclusions Specific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host’s undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced

Effect of vibration on osteoblastic and osteoclastic activities: Analysis of bone metabolism using goldfish scale as a model for bone

Science.gov (United States)

Suzuki, N.; Kitamura, K.; Nemoto, T.; Shimizu, N.; Wada, S.; Kondo, T.; Tabata, M. J.; Sodeyama, F.; Ijiri, K.; Hattori, A.

In osteoclastic activity during space flight as well as hind limb unloading by tail suspension, inconsistent results have been reported in an in vivo study. The bone matrix plays an important role in the response to physical stress. However, there is no suitable in vitro co-culture system of osteoblasts and osteoclasts including bone matrix. On the other hand, fish scale is a calcified tissue that contains osteoblasts, osteoclasts, and bone matrix, all of which are similar to those found in human bones. Recently, we developed a new in vitro model system using goldfish scale. This system can detect the activities of osteoclasts and osteoblasts with tartrate-resistant acid phosphatase and alkaline phosphatase as the respective markers and precisely analyze the co-relationship between osteoblasts and osteoclasts. Using this system, we analyzed the bone metabolism under various degrees of acceleration (0.5-, 1-, 2-, 4-, and 6-G) by vibration with a G-load apparatus. After loading for 5 and 10 min, the scales were incubated for 6 and 24 h. The osteoblastic and osteoclastic activities were then measured. The osteoblastic activities gradually increased corresponding to 1-G to 6-G acceleration. In addition, ER mRNA expression was the highest under 6-G acceleration. On the other hand, the osteoclastic activity decreased at 24 h of incubation under low acceleration (0.5- and 1-G). This change coincided with TRAP mRNA expression. Under 2-G acceleration, the strength of suppression in osteoclastic activity was the highest. The strength of the inhibitory action under 4- and 6-G acceleration was lower than that under 2-G acceleration. In our co-culture system, osteoblasts and osteoclasts in the scale sensitively responded to several degrees of acceleration. Therefore, we strongly believe that our in vitro co-culture system is useful for the analysis of bone metabolism under loading or unloading.

Use of bone marrow derived stem cells in a fracture non-union

Directory of Open Access Journals (Sweden)

Binod C. Raulo

2012-01-01

Full Text Available This is an attempt of using in vitro cultured mesenchymal stem cells (MSCs from bone marrow in joining of a fracture non-union. Bone marrow cells were obtained and differentially centrifuged for MSCs that were grown in vitro in mesenchymal stem cell basal medium aseptically, for 10 d. The cell mass was injected around the fracture non-union. Healthy conditions of development of tissue regeneration at the trauma site and due bone joining were recorded. It is concluded that in vitro cultured MSCs had a blithesome effect on the fracture non-union.

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

Science.gov (United States)

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

Bone--bone marrow interface (endosteum) potential relationship of microenvironments in the regulation of response to internal emitters

International Nuclear Information System (INIS)

Wilson, F.D.; Pool, R.R.; Stitzel, K.; Momeni, M.H.

1976-01-01

The interface between bone and bone marrow is examined in relation to radiation effects, with attention to new concepts of hematopoiesis. Such concepts propose a functional role of stroma in regulating the commitment of pluripotent stem cells as well as in the production of colony stimulating activity (CSA) including candidate granulopoietin(s). Morphologic examples are included, underlining the concept that stroma (including bone) and hematopoietic elements respond as a functional unit to injury to marrow elements. The methylcellulose bone marrow culture system is reviewed as it may relate to a method for quantitation of hematopoietic colonies (CFU-C), humoral regulators for granulopoiesis (CSA), and potentially as a method of quantitating mesenchymal progenitor populations (PFU-C). Based on these and other observations cited, a model depicting a tentative positioning of cells at risk relative to bone-seeking radionuclides is presented

Cell kinetics of differentiation of Na+-dependent hexose transport in a cultured renal epithelial cell line

International Nuclear Information System (INIS)

Cook, J.S.; Weiss, E.R.

1985-01-01

Fully differentiated cells of the renal proximal tubule have the capability of taking up hexoses across their apical borders by transport coupled to the Na + -electrochemical gradient. This property is also found in postconfluent cultures of the cloned cell line LLC-PK 1 , a morphologically polarized line of renal cells. Postconfluent cells develop the Na + -dependent capacity to transport hexoses at their apical surface. This function is not observable during the growth phase of the cultures. To analyze the developmental process at the cellular level a method has been derived to separate transporting cells, expressing the differentiated function, from nontransporting cells. The method is based on the swelling of the cells accompanying the uptake of the nonmetabolizable glucose analog alpha methylglucoside. The swollen cells have a lower buoyant density than the undifferentiated cells and may be separated from them on density gradients. Analysis of the distribution of cells on such gradients shows that after the cells reach confluence the undifferentiated subpopulation is recruited onto the differentiation pathway with a rate constant of 0.2 per day, that 5 to 7 days are required for a cell to traverse this pathway to the fully differentiated state, and that once the maximum uptake capacity is achieved the cells do not develop further

Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds

DEFF Research Database (Denmark)

Bjerre, Lea; Bünger, Cody E; Kassem, Moustapha

2008-01-01

Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate......-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity...... assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone...

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

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Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Polyethylene and methyl methacrylate particle-stimulated inflammatory tissue and macrophages up-regulate bone resorption in a murine neonatal calvaria in vitro organ system.

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Ren, Weiping; Wu, Bin; Mayton, Lois; Wooley, Paul H

2002-09-01

There is considerable evidence that orthopaedic wear debris plays a crucial role in the pathology of aseptic loosening of joint prostheses. This study examined the effect of inflammatory membranes stimulated with methyl methacrylate and polyethylene on bone resorption, using the murine air pouch model. The capacity of RAW 264.7 mouse macrophages exposed to polymer particles to produce factors affecting bone metabolism was also studied. Neonatal calvaria bones were co-cultured with either pouch membranes or conditioned media from activated macrophages. Bone resorption was measured by the release of calcium from cultured bones, and the activity of tartrate-resistant acid phosphatase in both bone sections and culture medium was also assayed. Results showed that inflammatory pouch membrane activated by methyl methacrylate and polyethylene enhanced osteoclastic bone resorption. Conditioned media from particles stimulated mouse macrophages also stimulated bone resorption, although this effect was weaker than resorption induced by inflammatory pouch membranes. The addition of the particles directly into the medium of cultured calvaria bones had little effect on bone resorption. Our observations indicate that both inflammatory tissue and macrophages provoked by particles can stimulate bone resorption in cultured mouse neonatal calvaria bones. This simple in vitro bone resorption system allows us to investigate the fundamental cellular and molecular mechanism of wear debris induced bone resorption and to screen potential therapeutic approaches for aseptic loosening.

MFH classification: differentiating undifferentiated pleomorphic sarcoma in the 21st Century.

Science.gov (United States)

Matushansky, Igor; Charytonowicz, Elizabeth; Mills, Joslyn; Siddiqi, Sara; Hricik, Todd; Cordon-Cardo, Carlos

2009-08-01

The essence and origin of malignant fibrous histiocytoma (MFH) have been debated for now close to five decades. Originally characterized as a morphologically unique soft-tissue sarcoma subtype of unclear etiology in 1963, with a following 15 years of research only to conclude that "the issue of histogenesis [of MFH] is largely unresolvable"; it is "now regarded as synonymous with [high grade] undifferentiated pleomorphic sarcoma and essentially represents a diagnosis of exclusion". Yet despite this apparent lack of progress, the first decade of the 21st century has seen some significant progress in terms of defining the origins of MFH. Perhaps more importantly these origins might also pave the way for novel therapies. This manuscript will highlight MFH's troubled history, discuss recent advances, and comment as to what the coming years may promise and what further needs to be done to make sure that progress continues.

CD133-expressing thyroid cancer cells are undifferentiated, radioresistant and survive radioiodide therapy

International Nuclear Information System (INIS)

Ke, Chien-Chih; Liu, Ren-Shyan; Yang, An-Hang; Liu, Ching-Sheng; Chi, Chin-Wen; Tseng, Ling-Ming; Tsai, Yi-Fan; Ho, Jennifer H.; Lee, Chen-Hsen; Lee, Oscar K.

2013-01-01

131 I therapy is regularly used following surgery as a part of thyroid cancer management. Despite an overall relatively good prognosis, recurrent or metastatic thyroid cancer is not rare. CD133-expressing cells have been shown to mark thyroid cancer stem cells that possess the characteristics of stem cells and have the ability to initiate tumours. However, no studies have addressed the influence of CD133-expressing cells on radioiodide therapy of the thyroid cancer. The aim of this study was to investigate whether CD133 + cells contribute to the radioresistance of thyroid cancer and thus potentiate future recurrence and metastasis. Thyroid cancer cell lines were analysed for CD133 expression, radiosensitivity and gene expression. The anaplastic thyroid cancer cell line ARO showed a higher percentage of CD133 + cells and higher radioresistance. After γ-irradiation of the cells, the CD133 + population was enriched due to the higher apoptotic rate of CD133 - cells. In vivo 131 I treatment of ARO tumour resulted in an elevated expression of CD133, Oct4, Nanog, Lin28 and Glut1 genes. After isolation, CD133 + cells exhibited higher radioresistance and higher expression of Oct4, Nanog, Sox2, Lin28 and Glut1 in the cell line or primarily cultured papillary thyroid cancer cells, and lower expression of various thyroid-specific genes, namely NIS, Tg, TPO, TSHR, TTF1 and Pax8. This study demonstrates the existence of CD133-expressing thyroid cancer cells which show a higher radioresistance and are in an undifferentiated status. These cells possess a greater potential to survive radiotherapy and may contribute to the recurrence of thyroid cancer. A future therapeutic approach for radioresistant thyroid cancer may focus on the selective eradication of CD133 + cells. (orig.)

Undifferentiated nasopharyngeal cancer (UCNT): current diagnostic and therapeutic aspects

International Nuclear Information System (INIS)

Altun, M.; Fandi, A.; Dupuis, O.; Cvitkovic, E.; Krajina, Z.; Eschwege, F.

1995-01-01

Undifferentiated carcinoma of the nasopharynx (UCNT) is a particular head and neck epidermoid lineage tumor related to the Epstein Barr Virus (EBV). It has geographically selective endemic epidemiologic features, without relation to external carcinogens. Its systemic aggressiveness is the source of most disease-related demises, because radiotherapy achieves excellent local control and a significant percentage of cure in patients with exclusive locoregional disease. Differences in the staging systems currently in use, the recent changes in imaging and radiotherapy technology, and the lack of distinction between UCNT and squamous cell carcinoma (SCC) of the nasopharynx in Western literature reports make for some difficulty in therapeutic results evaluation when analyzing available literature. Its chemosensitivity is a relatively recent acknowledged fact, and its use in metastatic patients results in a high percentage of objective responses, many of long duration. Neoadjuvant cisplatin-based chemotherapy seems to be of benefit, but outstanding controversies in this regard will be soon answered through ongoing phase III trials. After a review of the current literature of all the above-mentioned aspects of this fascinating nosologic entity, our own experience, both in metastatic and locoregional disease patients is analyzed

Infections and inflammations of bones

International Nuclear Information System (INIS)

Rogers, L.F.

1987-01-01

Infections of bone have been conveniently divided into three categories reflecting the source of the infection: (1) hematogenous osteomyelitis, (2) implantation osteomyelitis caused by bacteria implanted or introduced with an open fracture, penetrating wound, or surgical procedure, and (3) secondary osteomyelitis with the bone involvement secondary to a continuous focus of soft-tissue infection related to peripheral vascular disease. In the series by Waldvogel and associates, hematogenous osteomyelitis accounted for only 19%, implantation osteomyelitis 47%, and secondary osteomyelitis related to vascular insufficiency 34% of all cases. Osteomyelitis may also be divided into acute, subacute, and chronic forms, dependent on the virulence of the organism, the response of the host, and the effectiveness of antibiotic treatment. Staphylococcus aureus is the most frequent offending organism. In children, in whom hematogenous infection is the rule, multiple foci of disease are relatively frequent, whereas in adults, the infection is usually limited to a single focus. The cause is usually established by obtaining a positive blood culture, a culture from an aspiration of the adjacent joint, or a direct aspiration of the involved bone or overlying soft tissues. Early recognition and treatment with antibiotics may minimize the radiographic findings of osteomyelitis

A facile in vitro model to study rapid mineralization in bone tissues.

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Deegan, Anthony J; Aydin, Halil M; Hu, Bin; Konduru, Sandeep; Kuiper, Jan Herman; Yang, Ying

2014-09-16

Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required. We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT). Two size aggregates (on average, large aggregates were 745 μm and small 79 μm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture. This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate

Undifferentiated embryonic cell transcription factor 1 (UTF1) and deleted in azoospermia-like (DAZL) expression in the testes of donkeys.

Science.gov (United States)

Lee, Y S; Jung, H J; Yoon, M J

2017-04-01

Putative markers for each specific germ cell stage can be a useful tool to study the fate and functions of these cells. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans, rats and horses. The deleted in azoospermia-like (DAZL) protein is also expressed by differentiated spermatogonia and primary spermatocytes in several species. However, whether the expression patterns of these molecular markers are identical and applicable to donkeys remains to be elucidated. The objective of this study was to investigate the expression patterns of UTF1 and DAZL in donkey testicular tissue, using immunohistochemistry (IHC). Testicular samples were collected from routine field castration of donkeys in Korea. The reproductive stages (pre- or post-puberty) of the testes were determined from the morphological characteristics of cross-sections of the seminiferous tubules. For IHC, the UTF1 and DAZL primary antibodies were diluted at 1:100 and 1:200, respectively. The immunolabelling revealed that UTF1 was expressed in approximately 50% of spermatogonia in the pre-pubertal stage, whereas its expression was limited to an early subset of spermatogonia in the post-pubertal stage. DAZL was expressed in some, but not all, spermatogonia in the pre-pubertal spermatogonia, and interestingly, its expression was also observed in spermatogonia and primary spermatocytes in the post-pubertal stage. Co-immunolabelling of the germ cells with both UTF1 and DAZL revealed three types of protein expression patterns at both reproductive stages, namely UTF1 only, DAZL only and both UTF1 and DAZL. These protein molecules were not expressed in Sertoli and Leydig cells. In conclusion, a co-immunolabelling system with UTF1 and DAZL antibodies may be used to identify undifferentiated (UTF1 only), differentiating (UTF1 and DAZL), and differentiated spermatogonia (DAZL only) in donkey testes. © 2017 Blackwell Verlag GmbH.

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

Science.gov (United States)

Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

2011-11-15

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

Translation into Portuguese, cross-cultural adaptation and validation of "The European Organization for Research and Treatment of Cancer-Quality of Life Questionnaire-Bone Metastases-22".

Science.gov (United States)

Miki-Rosário, Natália; Garcia Filho, Reynaldo Jesus; Garcia, Jairo Greco; Dini, Gal Moreira; Bottomley, Andrew; Chow, Edward; Sabino Neto, Miguel

2016-07-01

The aim of the present study was to conduct a cross-cultural adaptation (with translation into Brazilian Portuguese) and validation of the European Organization for Research and Treatment of Cancer-Quality of Life Questionnaire-Bone Metastases-22 (EORTC QLQ-BM22). Ninety-five bone metastasis patients (31 men and 64 women, mean age 58.36±8.90 years) took part in the investigation. The translation guide of the EORTC was used to translate from English into Brazilian Portuguese and adapt the instrument culturally. The reliability and the face, content and construct validities were tested. Internal consistency was estimated using Cronbach's alpha for the total score, pain and functional subscales of the EORTC QLQ-BM22 (0.93, 0.86, 0.90). Reliability was analyzed by Pearson's correlation and intraclass correlation coefficients (ICCs). The correlations were higher than the recommended value of 0.75, which indicated good test-retest reliability. Construct validity was demonstrated by correlation with the questionnaire medical outcome study questionnaire 36-Item Short Form Survey (SF-36). It showed significant correlation between the fields of QLQ-BM22 and the SF-36 (P≤0.001). The EORTC QLQ-BM22 was translated into Brazilian Portuguese, was culturally adapted and was proven to be reliable, with face, content and construct validity.

Comparative efficacy of tulathromycin versus a combination of florfenicol-oxytetracycline in the treatment of undifferentiated respiratory disease in large numbers of sheep

Directory of Open Access Journals (Sweden)

Mohsen Champour

2015-09-01

Full Text Available The objective of this study was to compare the efficacy of tulathromycin (TUL with a combination of florfenicol (FFC and long-acting oxytetracycline (LAOTC in the treatment of naturally occurring undifferentiated respiratory diseases in large numbers of sheep. In this study, seven natural outbreaks of sheep pneumonia in Garmsar, Iran were considered. From these outbreaks, 400 sheep exhibiting the signs of respiratory diseases were selected, and the sheep were randomly divided into two equal groups. The first group was treated with a single injection of TUL (dosed at 2.5 mg/kg body weight, and the second group was treated with concurrent injections of FFC (dosed at 40 mg/kg bwt and LAOTC (dosed at 20 mg/kg bwt. In the first group, 186 (93% sheep were found to be cured 5 days after the injection, and 14 (7% sheep needed further treatment, of which 6 (3% were cured, and 8 (4% died. In the second group, 172 (86% sheep were cured after the injections, but 28 (14% sheep needed further treatment, of which 10 (5% were cured, and 18 (9% died. This study revealed that TUL was more efficacious as compared to the combined treatment using FFC and LAOTC. As the first report, this field trial describes the successful treatment of undifferentiated respiratory diseases in large numbers of sheep. Thus, TUL can be used for the treatment of undifferentiated respiratory diseases in sheep. [J Adv Vet Anim Res 2015; 2(3.000: 279-284

Establishment of a new model for culturing rabbit osteoblasts in vitro

International Nuclear Information System (INIS)

Cao Xianying; Yin Meizhen; Zhang Lina; Li Shipu; Cao Yang

2006-01-01

To establish an experimental model for culturing rabbit osteoblasts in vitro, the osteoblasts were isolated from the calvarial bone of a 15-day old rabbit using a method of culturing the bone pieces in a medium after they had been digested by an enzyme for 15 min. The acquired cells were assayed by cell morphology, alkaline phosphatase activity and production of a mineralized matrix. The results showed that the cells had the morphologic characteristics and some biological behaviours of osteoblasts. Based on the primary isolation of osteoblasts from bone and combining digestion with explants, a novel model for culturing rabbit osteoblasts in vitro was established, which is easy, efficient and effective. This model can be used in many studies of osteogenesis mechanisms and bone replacement materials. (communication)

Bone conditioned media (BCM) improves osteoblast adhesion and differentiation on collagen barrier membranes.

Science.gov (United States)

Fujioka-Kobayashi, Masako; Caballé-Serrano, Jordi; Bosshardt, Dieter D; Gruber, Reinhard; Buser, Daniel; Miron, Richard J

2016-07-04

The use of autogenous bone chips during guided bone regeneration procedures has remained the gold standard for bone grafting due to its excellent combination of osteoconduction, osteoinduction and osteogenesis. Recent protocols established by our group have characterized specific growth factors and cytokines released from autogenous bone that have the potential to be harvested and isolated into bone conditioned media (BCM). Due to the advantageous osteo-promotive properties of BCM, the aims of the present study was to pre-coat collagen barrier membranes with BCM and investigate its effect on osteoblast adhesion, proliferation and differentiation for possible future clinical use. Scanning electron microscopy (SEM) was first used to qualitative assess BCM protein accumulation on the surface of collagen membranes. Thereafter, undifferentiated mouse ST2 stromal bone marrow cells were seeded onto BioGide porcine derived collagen barrier membranes (control) or barrier membranes pre-coated with BCM (test group). Control and BCM samples were compared for cell adhesion at 8 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 5 days for osteoblast differentiation markers including Runx2, alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Mineralization was further assessed with alizarin red staining at 14 days post seeding. SEM images demonstrated evidence of accumulated proteins found on the surface of collagen membranes following coating with BCM. Analysis of total cell numbers revealed that the additional pre-coating with BCM markedly increased cell attachment over 4 fold when compared to cells seeded on barrier membranes alone. No significant difference could be observed for cell proliferation at all time points. BCM significantly increased mRNA levels of osteoblast differentiation markers including ALP, OCN and BSP at 5 days post seeding. Furthermore, barrier membranes pre-coated with BCM demonstrated a 5-fold increase in alizarin

Recovery of the proliferative and functional integrity of mouse bone marrow in long-term cultures established after whole-body irradiation at different doses and dose rates

International Nuclear Information System (INIS)

Bierkens, J.G.; Hendry, J.H.; Testa, N.G.

1991-01-01

Injury inflicted upon the bone marrow stroma following whole-body irradiation and its repair over a 1-year period has been assessed in murine long-term bone marrow cultures established at increasing time intervals after irradiation. Different doses at different dose rates (10 Gy at 0.05 cGy/min, 4.5 Gy and 10 Gy at 1.6 cGy/min, and 4 x 4.5 Gy [3 weeks between doses] at 60 cGy/min) were chosen so as to maximize differences in effect in the stroma. The cellularity of the adherent layer in long-term cultures established 1 month after irradiation was reduced by 40%-90% depending on the dose and dose rate. Simultaneous with the poor ability of the marrow to form adherent layers, the cumulative spleen colony-forming unit (CFU-S) and granulocyte-macrophage colony-forming cell (GM-CFC) production over a 7-week period was reduced to 0% and 30% of control cultures, respectively. The slow recovery of the adherent layer was paralleled by an increase in the numbers of CFU-S and GM-CFC in the supernatant. Cultures established from repeatedly irradiated mice performed poorly over the entire 1-year period. Whereas the regeneration of the stroma was near complete 1 year after irradiation, the CFU-S and GM-CFC levels reached only between 50% and 80% of control cultures, respectively. Also, the concentration of CFU-S and GM-CFC in the supernatant remained persistently lower in cultures established from irradiated mice as compared to control cultures. The levels of sulfated glycosaminoglycans, which have been implicated in the establishment of the functional integrity of the microenvironment, were not reduced in the adherent layers at any time after irradiation. These results indicate that the regeneration of the stroma is accompanied by an incomplete recovery of active hemopoiesis in vitro

Study of acute undifferentiated fever cases and their etiologies in rural Konkan area of Maharashtra state

Directory of Open Access Journals (Sweden)

Patil S. N

2016-08-01

Full Text Available Background: Acute undifferentiated fever (AUF is a common cause for which the patients seek health care in India. It is region specific and has similar clinical presentation, with varied etiologies. Due to this it posses challenge to the diagnosis, treatment and public health. Majority of patients present with nondescript symptoms. Scrub typhus, Malaria, Enteric Fever, Dengue, Leptospirosis, Chikungunya, Spotted fever, Rickettsiosis, Hantavirus, Q fever, Brucellosis, Influenza and other bacterial infections are some of the common etiologies of AUF. The prevalence of local AUF etiologies helps to prioritize differential diagnosis and guide the treatment. The study aimed to find out the predominant AUF etiologies in the rural Konkan area of Maharashtra state in India. Materials and Methods: This prospective observational study was conducted at a tertiary care hospital on the samples received from District hospitals and Primary health centers from Sindhudurg District of Maharashtra state for the duration of October 2012 to January 2014. Patients with age 5years and with classical symptoms of febrile illness were included in the study. About 500 blood samples received were investigated for Malaria, Bacterial culture sensitivity, Leptospira culture, ELISA for scrub typhus, Brucella, Dengue and Leptospira and further evaluated for commonest region specific AUF etiology. Results: The study included 500 blood samples obtained from patients presenting with classical symptoms of AUF. Samples received from males showed highest number of positive cases amounting for 82.47% with majority of cases (83% cases in middle age group. The sero-positivity of samples accounted for 42.8%. Brucella was the most common cause of AUF (28.50% followed by Leptospira (27.10% and Scrub typhus (21.49%. Interestingly there were no positive cases of malaria and only 11.21% samples positive for Dengue which are considered as most common AUF etiologies and treated accordingly

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture

Energy Technology Data Exchange (ETDEWEB)

Perestrelo, Ana Rubina [Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve (Portugal); IBB - Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), Universidade do Algarve (Portugal); PhD Program in Biomedical Sciences, Universidade do Algarve (Portugal); Grenha, Ana [IBB - Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), Universidade do Algarve (Portugal); Rosa da Costa, Ana M. [Centro de Investigação em Química do Algarve (CIQA) and Departamento de Química e Farmácia, Faculdade de Ciências e Tecnologia, Universidade do Algarve (Portugal); Belo, José António, E-mail: jose.belo@fcm.unl.pt [Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve (Portugal); IBB - Institute for Biotechnology and Bioengineering, Centre for Molecular and Structural Biomedicine (CBME), Universidade do Algarve (Portugal); Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa (Portugal)

2014-07-01

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture

International Nuclear Information System (INIS)

Perestrelo, Ana Rubina; Grenha, Ana; Rosa da Costa, Ana M.; Belo, José António

2014-01-01

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro

Foetal stem cell derivation & characterization for osteogenic lineage

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A Mangala Gowri

2013-01-01

Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

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Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

2017-06-01

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

LENUS (Irish Health Repository)

Farrell, Eric

2011-01-31

Abstract Background Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients\\' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. Methods MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. Results After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. Conclusions The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of �

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

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Mandatori, Domitilla; Penolazzi, Letizia; Pipino, Caterina; Di Tomo, Pamela; Di Silvestre, Sara; Di Pietro, Natalia; Trevisani, Sara; Angelozzi, Marco; Ucci, Mariangela; Piva, Roberta; Pandolfi, Assunta

2018-02-01

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment. Copyright © 2017 John Wiley & Sons, Ltd.

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

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

2016-03-01

functionalized with different amounts of quercetin and obtain new composite materials which display both the good bioactivity of the inorganic phase and the therapeutic properties of the flavonoid. The innovative in vitro model developed in this study, which involves co-culture of osteoblast, osteoclast and endothelial cells, allows to state that the new materials exert a beneficial action onto bone repair microenvironment, stimulating osteoblast proliferation and activity, downregulating osteoclastogenesis, and supporting microangiogenetic processes necessary for new bone formation. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

New strategies for the treatment of undifferentiated thyroid cancer and poorly differentiated thyroid cancer

International Nuclear Information System (INIS)

Juvenal, Guillermo J.

2006-01-01

Undifferentiated thyroid cancer, which accounts for about 5-10% of thyroid cancer cases, is a very aggressive tumor with no effective treatment, since it lacks iodine uptake and does not respond to radio or chemotherapy. The prognosis of these patients is bad, due to the rapid growth of the tumor and the early development of metastasis. Oncogenes and tumor suppressor genes are involved in the genetic changes that underlie thyroid cancer, as all kinds of tumors. The characterization of these proteins is being exploited to delineate new therapeutic strategies for the treatment of this cancer. This work is focused on those compounds or therapeutic approaches that are being used in clinical essays or in animal models. (author) [es

Effects of vitamin D metabolites on cellular Ca2+ and on Ca transport in primary cultures of bone cells.

Science.gov (United States)

Eilam, Y; Szydel, N; Harell, A

1980-09-01

Both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) exerted direct effects on Ca2+ transport and accumulation in primary cultures of bone cells. The following changes were recorded. (1) A significant decrease in the amount of intracellular exchangeable Ca2+. (2) A marked increase in the rate constants of efflux from the 'slow'-turnover intracellular Ca pool. (3) A marked increase in the 'initial rate' of Ca influx into the cells. Thus, vitamin D metabolites caused an increase in the turnover of Ca2+ in bone cells and altered the steady-stae level of intracellular exchangeable Ca2+. Whereas the changes in the rate of efflux were abolished in the presence of inhibitors of protein synthesis, the increase in the rate of influx was not sensitive to these inhibitors. It is suggested that the changes in the two fluxes were mediated by different mechanisms and that the changes in influx were due to a direct effect of vitamin D metabolites on the cellular membranes.

Blocking the expression of both bone sialoprotein (BSP) and osteopontin (OPN) impairs the anabolic action of PTH in mouse calvaria bone.

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Bouleftour, Wafa; Bouet, Guenaelle; Granito, Renata Neves; Thomas, Mireille; Linossier, Marie-Thérèse; Vanden-Bossche, Arnaud; Aubin, Jane E; Lafage-Proust, Marie-Hélène; Vico, Laurence; Malaval, Luc

2015-03-01

Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1-84 (iPTH) over the calvaria of BSP knockout (-/-) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and -/- mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from -/- mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5× higher in -/- versus +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in -/- cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP +/+ calvaria, while it blunted iPTH effects in -/- mice, reduced to a modest increase in periosteum thickness. In -/- (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins. © 2014 Wiley Periodicals, Inc., A Wiley Company.

In Vivo Bone Formation Within Engineered Hydroxyapatite Scaffolds in a Sheep Model.

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Lovati, A B; Lopa, S; Recordati, C; Talò, G; Turrisi, C; Bottagisio, M; Losa, M; Scanziani, E; Moretti, M

2016-08-01

Large bone defects still represent a major burden in orthopedics, requiring bone-graft implantation to promote the bone repair. Along with autografts that currently represent the gold standard for complicated fracture repair, the bone tissue engineering offers a promising alternative strategy combining bone-graft substitutes with osteoprogenitor cells able to support the bone tissue ingrowth within the implant. Hence, the optimization of cell loading and distribution within osteoconductive scaffolds is mandatory to support a successful bone formation within the scaffold pores. With this purpose, we engineered constructs by seeding and culturing autologous, osteodifferentiated bone marrow mesenchymal stem cells within hydroxyapatite (HA)-based grafts by means of a perfusion bioreactor to enhance the in vivo implant-bone osseointegration in an ovine model. Specifically, we compared the engineered constructs in two different anatomical bone sites, tibia, and femur, compared with cell-free or static cell-loaded scaffolds. After 2 and 4 months, the bone formation and the scaffold osseointegration were assessed by micro-CT and histological analyses. The results demonstrated the capability of the acellular HA-based grafts to determine an implant-bone osseointegration similar to that of statically or dynamically cultured grafts. Our study demonstrated that the tibia is characterized by a lower bone repair capability compared to femur, in which the contribution of transplanted cells is not crucial to enhance the bone-implant osseointegration. Indeed, only in tibia, the dynamic cell-loaded implants performed slightly better than the cell-free or static cell-loaded grafts, indicating that this is a valid approach to sustain the bone deposition and osseointegration in disadvantaged anatomical sites.

In vitro prostaglandin E2 stimulation of 45Ca mobilization from chick bone

International Nuclear Information System (INIS)

Satterlee, D.G.; Amborski, G.F.; McIntyre, M.D.; Parker, M.S.; Jacobs-Perry, L.A.

1984-01-01

The ability of prostaglandin E2 (PGE2) to mobilize 45 Ca from chick embryo long bones was assayed in an in vitro bone culture system. Concentrations of PGE2 tested ranged from 10(-9) to 10(-5) M. The PGE2 was effective in stimulating release of 45 Ca from prelabelled bones at all concentrations tested except at 10(-9) M. In addition, stimulation of 45 Ca release could be produced daily for 4 consecutive days of PGE2 culture-pulsing at what appeared to be the optimal PGE2 concentration, 10(-7) M. The authors conclude, as in mammals, PGE2 is a potent stimulator of calcium mobilization from avian bone. The potential involvement of prostaglandins in eggshell formation is discussed

Influence of diphenylhydantoin on lysosomal enzyme release during bone resorption in vitro

International Nuclear Information System (INIS)

Lerner, U.; Haenstroem, L.

1980-01-01

The effect of diphenylhydantoin (DPH) on the release of lysosomal enzymes during resorption of cultured mouse calvarial bone was studied. The enzyme activities of β-glucuronidase and β-galactosidase in the culture medium was taken as indicators for lysosomal enzyme release. In concentrations 50 μg/ml or higher, DPH inhibited the release of β-glucuronidase and β-galactosidase in parallel with bone resorption as indicated by reduced release of 4 Ca, Ca 2 , Psub(i) and hydroxyproline. The release of the cytosolic enzyme lactate dehydrogenase was not influenced by concentrations of DPH up to 50 μg/ml but higher concentrations caused an increased release indicating cell injury. When bone resorption was stimulated by prostaglandin E 2 , DPH(50 μg/ml) also reduced the mobilization of bone mineral and the release of β- glucuronidase without influencing the release of lactate dehydrogenase. It is suggested that DPH by interfering with cellular release processes reduces the resorption on bone. (author)

Radiation Safety Culture in Medicine AFROSAFE_R_A_D

International Nuclear Information System (INIS)

Nyabanda, R.

2017-01-01

Ionizing radiation that include X-rays and Gamma rays Radio waves, infrared and visible light carries sufficient energy to free electrons from atoms or molecules. Becquerel first person to discover evidence of radioactivity, who shared a Nobel Prize for physics in 1903 with Marie and Pierre Curie. Prof Sievert and Louis Harold Gray are the Medical physicists who had major contribution in the study of the biological effects of radiation. Ionizing radiation causes displacement of an electron which can inflict damage on DNA either directly or indirectly. A radiation-safety campaign developed by the radiation health workers in Africa. Radiosensitive organs is highest in cells which are highly mitotic or undifferentiated. E.g basal epidermis, bone marrow, thymus, gonads, and lens cells. Relatively low radiosensitivity in muscle, bones, and nervous system tissues. A radiation-safety campaign developed by the radiation health workers in Africa. AFROSAFE Strategies Strengthen radiation protection of patients, health workers and public, Promote safe and appropriate use of ionizing radiation in medicine. Foster improvement of the benefit-risk dialogue with patients and the public. Enhance the safety and quality of radiological procedures in medicine, Promote safety in radiological equipment and facilities and Promote research in radiation protection and safety

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

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Chang Qing

2011-04-01

Full Text Available Chondrocytes and bone marrow mesenchymal stem cells (BMSCs are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs. This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

Science.gov (United States)

Qing, Chang; Wei-ding, Cui; Wei-min, Fan

2011-04-01

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Autologous bone marrow purging with LAK cells.

Science.gov (United States)

Giuliodori, L; Moretti, L; Stramigioli, S; Luchetti, F; Annibali, G M; Baldi, A

1993-12-01

In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation.

Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

Directory of Open Access Journals (Sweden)

Mehdi Forouzandeh-Moghadam

2009-01-01

Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

Chinese red yeast rice (Monascus purpureus-fermented rice promotes bone formation

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Rabie Bakr

2008-03-01

Full Text Available Abstract Background Statin can induce the gene expression of bone morphogenetic protein-2. Red yeast rice (RYR, Hongqu, i.e. rice fermented with Monascus purpureus, contains a natural form of statin. This study demonstrates the effects of RYR extract on bone formation. Methods Bone defects were created in the parietal bones of two New Zealand white rabbits. In the test animal, two defects were grafted with collagen matrix mixed with RYR extract. In the control animal, two defects were grafted with collagen matrix alone. UMR 106 cell line was used to test RYR extract in vitro. In the control group, cells were cultured for three durations (24 hours, 48 hours and 72 hours without any intervention. In the RYR group, cells were cultured for the same durations with various concentrations of RYR extract (0.001 g/ml, 0.005 g/ml and 0.01 g/ml. Bicinchoninic acid (BCA assay, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and alkaline phosphatase (ALP assay were performed to measure total protein, mitochondrial activity and bone cell formation respectively. Results The test animal showed more formation of new bone in the defects than the control animal. RYR significantly increased the optical density in the MTT assay and ALP activity in vitro. Conclusion RYR extract stimulated new bone formation in bone defects in vivo and increased bone cell formation in vitro.

The CIPRUS study, a nurse-led psychological treatment for patients with undifferentiated somatoform disorder in primary care: study protocol for a randomised controlled trial

NARCIS (Netherlands)

Sitnikova, Kate; Leone, Stephanie S.; Zonneveld, Lyonne N. L.; van Marwijk, Harm W. J.; Bosmans, Judith E.; van der Wouden, Johannes C.; van der Horst, Henriëtte E.

2017-01-01

Background: Up to a third of patients presenting medically unexplained physical symptoms in primary care may have a somatoform disorder, of which undifferentiated somatoform disorder (USD) is the most common type. Psychological interventions can reduce symptoms associated with USD and improve

The CIPRUS study, a nurse-led psychological treatment for patients with undifferentiated somatoform disorder in primary care : study protocol for a randomised controlled trial

NARCIS (Netherlands)

Sitnikova, Kate; Leone, Stephanie S; Zonneveld, Lyonne N L; van Marwijk, Harm W J; Bosmans, Judith E; van der Wouden, Johannes C; van der Horst, Henriëtte E

2017-01-01

BACKGROUND: Up to a third of patients presenting medically unexplained physical symptoms in primary care may have a somatoform disorder, of which undifferentiated somatoform disorder (USD) is the most common type. Psychological interventions can reduce symptoms associated with USD and improve

Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

Science.gov (United States)

Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

2016-02-01

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

Resorption behavior of a nanostructured bone substitute: in vitro investigation and clinical application.

Science.gov (United States)

Reichert, Christoph; Götz, Werner; Reimann, Susanne; Keilig, Ludger; Hagner, Martin; Bourauel, Christoph; Jäger, Andreas

2013-03-01

To develop an in vitro assay for quantitative analysis of the degradation to which a bone substitute is exposed by osteoclasts. The aim of establishing this method was to improve the predictability of carrying out tooth movements via bone substitutes and to provide a basis for verification in exemplary clinical cases. After populating a bone substitute (NanoBone®; ArtOss, Germany) with osteoclastic cells, inductively-coupled mass spectrometry was used to evaluate changing calcium levels in the culture medium as a marker of resorption activity. It was observed that calcium levels increased substantially in the culture medium with the cells populating the bone substitute. This in vitro assay is a valid method that can assist clinicians in selecting the appropriate materials for certain patients. While tooth movements occurring through this material were successful, uncertainty about the approach will remain as long-term results are not available.

Influence on bone metabolism of dietary trace elements, protein, fat, carbohydrates, and vitamins.

Science.gov (United States)

Sarazin, M; Alexandre, C; Thomas, T

2000-01-01

Osteoporosis is a multifactorial disease driven primarily by the genetic factors that control bone metabolism. Among environmental factors, diet may play a key role, affording a target for low-cost intervention. Calcium and vitamin D are well known to affect bone metabolism. Other nutrients may influence bone mass changes; for instance, a number of trace elements and vitamins other than vitamin D are essential to many of the steps of bone metabolism. A wide variety of foods provide these nutrients, and in industrialized countries deficiencies are more often due to idiosyncratic eating habits than to cultural influences. Both culture and vogue influence the amount of carbohydrate, fat, and protein in the typical diet. In children, the current trend is to reduce protein and to increase carbohydrate and fat. Data from epidemiological and animal studies suggest that this may adversely affect bone mass and the fracture risk.

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

International Nuclear Information System (INIS)

Perkins, S.; Fleischman, R.A.

1988-01-01

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

Bone tissue engineering on amorphous carbonated apatite and crystalline octacalcium phosphate-coated titanium discs

NARCIS (Netherlands)

Dekker, Robert J.; de Bruijn, Joost Dick; Stigter, Martin; Barrère, F.; Layrolle, Pierre; van Blitterswijk, Clemens

2005-01-01

Poor fixation of bone replacement implants, e.g. the artificial hip, in implantation sites with inferior bone quality and quantity may be overcome by the use of implants coated with a cultured living bone equivalent. In this study, we tested, respectively, amorphous carbonated apatite (CA)- and

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

International Nuclear Information System (INIS)

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

1991-01-01

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

Comparison of fracture site callus with iliac crest bone marrow as the source of plastic-adherent cells

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Achmad Zaki

2013-05-01

Full Text Available Background: Red marrow has been described as the main source of mesenchymal stem cells although its aspiration and isolation from bone marrow was reported to have significant donor site morbidity. Since secondary bone healing occurs through formation of callus as the result of proliferation and differentiation of mesenchymal stem cells, callus may become alternative source for mesenchymal stem cells. In this study, we compared the number of plastic-adherent cells from fracture site callus and bone marrow of iliac crest after two and four weeks of culture.Methods: Sixteen New Zealand rabbits were fracturized at the femoral shaft. Then, these rabbits were taken care. After two weeks of fracturization, 3 mL iliac crest bone marrow aspiration and callus extraction of eight rabbits were cultured (group I. The other eight rabbits were treated equally after four weeks of fracturization (group II. Simultaneously, the cultures were observed after one and two weeks. Four weeks later, they were harvested. Cells were counted using Neubauer hemocytometer. The average number of cells between the sources and groups were statistically analyzed using the unpaired t-test. Results: In group I, there were 2.6 ± 0.1 x 104 cells in the culture of iliac crest bone marrow aspirate and 2.5 ± 0.1 x 104 cells in culture of callus extract from fracture site (p = 0.34. In group II, there were 2.7 ± 0.1 x 104 cells and 2.1 ± 0.1 x 104 cells, respectively (p < 0.001.Conclusion: Fracture site callus at the second week post-fracturization may be potential as source of plastic-adherent cells compared with iliac crest bone marrow. (Med J Indones. 2013;22:70-5Keywords: Bone marrow, fracture site callus, iliac crest, long bone, mesenchymal stem cell, plastic-adherent cells

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

Science.gov (United States)

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.

In the trail of a new bio-sensor for measuring strain in bone: osteoblastic biocompatibility.

Science.gov (United States)

Carvalho, Lídia; Alberto, Nélia J; Gomes, Pedro S; Nogueira, Rogério N; Pinto, João L; Fernandes, Maria H

2011-06-15

Fibre Bragg Grating (FBG) is an optical sensor recorded within the core of a standard optical fibre, which responds faithfully to strain and temperature. FBG sensors are a promising alternative to other sensing methodologies to assess bone mechanics in vivo. However, response of bone cells/bone tissue to FBGs and its sensing capability in this environment have not been recorded yet. The present study addressed these issues in long-term human osteoblastic cell cultures. Results showed that osteoblastic cells were able to adhere and proliferate over the fibre and, also, the protective polymer coating. RT-PCR analysis showed the expression of Col I, ALP, BMP-2, M-CSF, RANKL and OPG. In addition, cultures presented high ALP activity and the formation of a calcium phosphate mineralized extracellular matrix. Cell behavior over the fibre without and with the coating polymer was similar to that found in cultures grown in standard tissue culture plates (control). In addition to the excellent osteoblastic cytocompatibility, FBGs maintained the physical integrity and functionality, as its sensing capability was not affected through the culture period. Results suggest the possibility of in vivo osseointegration of the optical fibre/FBGs anticipating a variety of applications in bone mechanical dynamics. Copyright © 2011 Elsevier B.V. All rights reserved.

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

Science.gov (United States)

Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C

2013-01-01

Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

Directory of Open Access Journals (Sweden)

Peter E Westerweel

Full Text Available Circulating Endothelial Progenitor Cell (EPC levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment.Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+Flk-1(+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+ hematopoietic progenitor cells (HPC and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed.In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro.EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Growing B Lymphocytes in a Three-Dimensional Culture System

Science.gov (United States)

Wu, J. H. David; Bottaro, Andrea

2010-01-01

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing

Comparison of the effects of stimulators and inhibitors of resorption on the release of lysosomal enzymes and radioactive calcium from fetal bone in organ culture

International Nuclear Information System (INIS)

Eilon, G.; Raisz, L.G.

1978-01-01

The release of lysosomal enzymes, collagenase, and previously incorporated 45 Ca from fetal rat long bones cultured in a chemically defined medium is compared. Parathyroid hormone (PTH) and prostaglandin E 2 increased the release of β-glucuronidase, acetylglucosaminidase, and cathepsin D, but showed little effect on collagenase activity in the medium at 48 h. The dose-response relations for β-glucuronidase and 45 Ca release were similar. However, the increase in lysosomal enzyme release was proportionally greater and occurred earlier than the increase in 45 Ca release. PTH also caused a significant increase in total β-glucuronidase activity in bone plus medium. Several agents which stimulate 45 Ca release at an optimal concentration, but not at a higher concentration, including dibutyryl cAMP, isobutylmethylxanthine, and the calcium ionophore, A23187, all increased lysosomal enzyme release at the concentration which increased 45 Ca release. Three inhibitors of bone resorption (calcitonin, cortisol, and colchicine) blocked lysosomal enzyme release at the same time that 45 Ca release decreased. When the bones escaped from calcitonin inhibition, both 45 Ca and lysosomalenzyme release increased. While colchicine blocked both lysosomal enzymes and 45 CA release, it actually increased the release of bone collagenase, and together with PTH or prostaglandin E 2 caused a large increase in free collagenase activity in the medium. These data indicate that lysosomal enzyme release is closely linked to bone resorption and suggest that lysosomal enzymes may have a primary role in initiating resorption, perhaps by acting on noncollagenous matrix or tissue components before mineral removal and collagen degradation

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

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Matthew M. Cook

2013-09-01

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

Effect of Modified Pectin Molecules on the Growth of Bone Cells

NARCIS (Netherlands)

Kokkonen, H.E.; Ilvesaro, J.M.; Morra, M.; Schols, H.A.; Tuukkanen, J.

2007-01-01

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

Energy Technology Data Exchange (ETDEWEB)

Fani, Nesa [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ziadlou, Reihane [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahhoseini, Maryam, E-mail: m.shahhoseini@royaninstitute.org [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Baghaban Eslaminejad, Mohamadreza, E-mail: eslami@royaninstitute.org [Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of)

2016-06-10

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

International Nuclear Information System (INIS)

Fani, Nesa; Ziadlou, Reihane; Shahhoseini, Maryam; Baghaban Eslaminejad, Mohamadreza

2016-01-01

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

DEFF Research Database (Denmark)

Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

2010-01-01

Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

Energy Technology Data Exchange (ETDEWEB)

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

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.

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

International Nuclear Information System (INIS)

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

2013-01-01

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

The effect of perfusion culture on proliferation and differentiation of human mesenchymal stem cells on biocorrodible bone replacement material

International Nuclear Information System (INIS)

Farack, J.; Wolf-Brandstetter, C.; Glorius, S.; Nies, B.; Standke, G.; Quadbeck, P.; Worch, H.; Scharnweber, D.

2011-01-01

Biocorrodible iron foams were coated with different calcium phosphate phases (CPP) to obtain a bioactive surface and controlled degradation. Further adhesion, proliferation and differentiation of SaOs-2 and human mesenchymal stem cells were investigated under both static and dynamic culture conditions. Hydroxyapatite (HA; [Ca 10 (PO 4 ) 6 OH 2 ]) coated foams released 500 μg/g iron per day for Dulbecco's modified eagle medium (DMEM) and 250 μg/g iron per day for McCoys, the unmodified reference 1000 μg/g iron per day for DMEM and 500 μg/g iron per day for McCoys, while no corrosion could be detected on brushite (CaHPO 4 ) coated foams. Using a perfusion culture system with conditions closer to the in vivo situation, cells proliferated and differentiated on iron foams coated with either brushite or HA while in static cell culture cells could proliferate only on Fe-brushite. We conclude that the degradation behaviour of biocorrodible iron foams can be varied by different calcium phosphate coatings, offering opportunities for design of novel bone implants. Further studies will focus on the influence of different modifications of iron foams on the expression of oxidative stress enzymes. Additional information about in vivo reactions and remodelling behaviour are expected from testing in implantation studies.

Global MicroRNA Profiling in Human Bone Marrow Skeletal—Stromal or Mesenchymal–Stem Cells Identified Candidates for Bone Regeneration

DEFF Research Database (Denmark)

Chang, Chi Chih; Venø, Morten T.; Chen, Li

2018-01-01

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem......RNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B...... cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of mi...

CXCR7 maintains osteosarcoma invasion after CXCR4 suppression in bone marrow microenvironment.

Science.gov (United States)

Han, Yan; Wu, Chunlei; Wang, Jing; Liu, Na

2017-05-01

The major cause of death in osteosarcoma is the invasion and metastasis. Better understanding of the molecular mechanism of osteosarcoma invasion is essential in developing effective tumor-suppressive therapies. Interaction between chemokine receptors plays a crucial role in regulating osteosarcoma invasion. Here, we investigated the relationship between CXCR7 and CXCR4 in osteosarcoma invasion induced by bone marrow microenvironment. Human bone marrow mesenchymal stem cells were co-cultured with osteosarcoma cells to mimic actual bone marrow microenvironment. Osteosarcoma cell invasion and CXCL12/CXCR4 activation were observed within this co-culture model. Interestingly, in this co-culture model, osteosarcoma cell invasion was not inhibited by suppressing CXCR4 expression with neutralizing antibody or specific inhibitor AMD3100. Downstream signaling extracellular signal-regulated kinase and signal transducer and activator of transcription 3 were not significantly affected by CXCR4 inhibition. However, suppressing CXCR4 led to CXCR7 upregulation. Constitutive expression of CXCR7 could maintain osteosarcoma cell invasion when CXCR4 was suppressed. Simultaneously, inhibiting CXCR4 and CXCR7 compromised osteosarcoma invasion in co-culture system and suppressed extracellular signal-regulated kinase and signal transducer and activator of transcription 3 signals. Moreover, bone marrow microenvironment, not CXCL12 alone, is required for CXCR7 activation after CXCR4 suppression. Taken together, suppressing CXCR4 is not enough to impede osteosarcoma invasion in bone marrow microenvironment since CXCR7 is activated to sustain invasion. Therefore, inhibiting both CXCR4 and CXCR7 could be a promising strategy in controlling osteosarcoma invasion.

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

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Jin H

2014-05-01

Full Text Available Han Jin,1 Kai Zhang,2 Chunyan Qiao,1 Anliang Yuan,1 Daowei Li,1 Liang Zhao,1 Ce Shi,1 Xiaowei Xu,1 Shilei Ni,1 Changyu Zheng,3 Xiaohua Liu,4 Bai Yang,2 Hongchen Sun11Department of Pathology, School of Stomatology, Jilin University, Changchun, People’s Republic of China; 2State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, People’s Republic of China; 3Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA; 4Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX, USAAbstract: 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(cDNA 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�

Age-associated bone loss and intraskeletal variability in the Imperial Romans.

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Cho, Helen; Stout, Sam Darrel

2011-01-01

An Imperial Roman sample from the Isola Sacra necropolis (100-300 A.D.) offered an opportunity to histologically examine bone loss and intraskeletal variability in an urban archaeological population. Rib and femur samples were analyzed for static indices of bone remodeling and measures of bone mass. The Imperial Romans experienced normal age-associated bone loss via increased intracortical porosity and endosteal expansion, with females exhibiting greater bone loss and bone turnover rates than in males. Life events such as menopause and lactation coupled with cultural attitudes and practices regarding gender and food may have led to increased bone loss in females. Remodeling dynamics differ between the rib and femur and the higher remodeling rates in the rib may be attributed to different effective age of the adult compacta or loading environment. This study demonstrates that combining multiple methodologies to examine bone loss is necessary to shed light on the biocultural factors that influence bone mass and bone loss.

Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining

DEFF Research Database (Denmark)

Kjærgaard, Kristian; Dreyer, Chris Halling; Ditzel, Nicholas

2016-01-01

expanded, adherent cells (A-CEAC). This study compares in vivo osteogenic capacity between A-CEAC and bone marrow derived culture expanded, adherent cells (BM-CEAC). Method. A-CEAC and BM-CEAC were isolated from five female sheep and seeded on hydroxyapatite granules prior to subcutaneous implantation...... in immunodeficient mice. The doses of cells in the implants were 0.5 × 106, 1.0 × 106, or 1.5 × 106 A-CEAC and 0.5 × 106 BM-CEAC, respectively. After eight weeks, bone volume versus total tissue volume (BV/TV) was quantified using histomorphometry. Origin of new bone was assessed using human vimentin (HVIM) antibody...... staining. Results. BM-CEAC yielded significantly higher BV/TV than any A-CEAC group, and differences between A-CEAC groups were not statistically significant. HVIM antibody stain was successfully used to identify sheep cells in this model. Conclusion. A-CEAC and BM-CEAC were capable of forming bone, and BM...

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Development of a Patient-Derived Xenograft (PDX of Breast Cancer Bone Metastasis in a Zebrafish Model

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Laura Mercatali

2016-08-01

Full Text Available Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231. The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT, revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient’s medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.

In Vitro Mimetic Models for the Bone-Cartilage Interface Regeneration.

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Bicho, Diana; Pina, Sandra; Oliveira, J Miguel; Reis, Rui L

2018-01-01

In embryonic development, pure cartilage structures are in the basis of bone-cartilage interfaces. Despite this fact, the mature bone and cartilage structures can vary greatly in composition and function. Nevertheless, they collaborate in the osteochondral region to create a smooth transition zone that supports the movements and forces resulting from the daily activities. In this sense, all the hierarchical organization is involved in the maintenance and reestablishment of the equilibrium in case of damage. Therefore, this interface has attracted a great deal of interest in order to understand the mechanisms of regeneration or disease progression in osteoarthritis. With that purpose, in vitro tissue models (either static or dynamic) have been studied. Static in vitro tissue models include monocultures, co-cultures, 3D cultures, and ex vivo cultures, mostly cultivated in flat surfaces, while dynamic models involve the use of bioreactors and microfluidic systems. The latter have emerged as alternatives to study the cellular interactions in a more authentic manner over some disadvantages of the static models. The current alternatives of in vitro mimetic models for bone-cartilage interface regeneration are overviewed and discussed herein.

Polyurethane foam scaffold as in vitro model for breast cancer bone metastasis.

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Angeloni, Valentina; Contessi, Nicola; De Marco, Cinzia; Bertoldi, Serena; Tanzi, Maria Cristina; Daidone, Maria Grazia; Farè, Silvia

2017-11-01

Breast cancer (BC) represents the most incident cancer case in women (29%), with high mortality rate. Bone metastasis occurs in 20-50% cases and, despite advances in BC research, the interactions between tumor cells and the metastatic microenvironment are still poorly understood. In vitro 3D models gained great interest in cancer research, thanks to the reproducibility, the 3D spatial cues and associated low costs, compared to in vivo and 2D in vitro models. In this study, we investigated the suitability of a poly-ether-urethane (PU) foam as 3D in vitro model to study the interactions between BC tumor-initiating cells and the bone microenvironment. PU foam open porosity (>70%) appeared suitable to mimic trabecular bone structure. The PU foam showed good mechanical properties under cyclic compression (E=69-109kPa), even if lower than human trabecular bone. The scaffold supported osteoblast SAOS-2 cell line proliferation, with no cytotoxic effects. Human adipose derived stem cells (ADSC) were cultured and differentiated into osteoblast lineage on the PU foam, as shown by alizarin red staining and RT-PCR, thus offering a bone biomimetic microenvironment to the further co-culture with BC derived tumor-initiating cells (MCFS). Tumor aggregates were observed after three weeks of co-culture by e-cadherin staining and SEM; modification in CaP distribution was identified by SEM-EDX and associated to the presence of tumor cells. In conclusion, we demonstrated the suitability of the PU foam to reproduce a bone biomimetic microenvironment, useful for the co-culture of human osteoblasts/BC tumor-initiating cells and to investigate their interaction. 3D in vitro models represent an outstanding alternative in the study of tumor metastases development, compared to traditional 2D in vitro cultures, which oversimplify the 3D tissue microenvironment, and in vivo studies, affected by low reproducibility and ethical issues. Several scaffold-based 3D in vitro models have been proposed

Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

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Park, Ji Eun; Park, Min Hee; Kim, Min Seong; Park, Yeo Reum; Yun, Jung Im; Cheong, Hee Tae; Kim, Minseok; Choi, Jung Hoon; Lee, Eunsong; Lee, Seung Tae

2017-12-01

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research. © 2017 International Federation for Cell Biology.

Response of bone marrow stromal cells to graded co-electrospun scaffolds and its implications for engineering the ligament-bone interface.

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Samavedi, Satyavrata; Guelcher, Scott A; Goldstein, Aaron S; Whittington, Abby R

2012-11-01

Biomaterial scaffolds with gradients in architecture, mechanical and chemical properties have the potential to improve the osseointegration of ligament grafts by recapitulating phenotypic gradients that exist at the natural ligament-bone (L-B) interface. Towards the larger goal of regenerating the L-B interface, this in vitro study was performed to investigate the potential of two scaffolds with mineral gradients in promoting a spatial gradient of osteoblastic differentiation. Specifically, the first graded scaffold was fabricated by co-electrospinning two polymer solutions (one doped with nano-hydroxyapatite particles) from offset spinnerets, while the second was created by immersing the first scaffold in a 5 × simulated body fluid. Rat bone marrow stromal cells, cultured in the presence of osteogenic supplements, were found to be metabolically active on all regions of both scaffolds after 1 and 7 days of culture. Gene expression of bone morphogenic protein-2 and osteopontin was elevated on mineral-containing regions as compared to regions without mineral, while the expression of alkaline phosphatase mRNA revealed the opposite trend. Finally, the presence of osteopontin and bone sialoprotein confirmed osteoblastic phenotypic maturation by day 28. This study indicates that co-electrospun scaffolds with gradients in mineral content can guide the formation of phenotypic gradients and may thus promote the regeneration of the L-B interface. Copyright © 2012 Elsevier Ltd. All rights reserved.

An antigen shared by human granulocytes, monocytes, marrow granulocyte precursors and leukemic blasts.

Science.gov (United States)

Shumak, K H; Rachkewich, R A

1983-01-01

An antibody to human granulocytes was raised in rabbits by immunization with granulocytes pretreated with rabbit antibody to contaminating antigens. The antibody reacted not only with granulocytes but also with monocytes and bone marrow granulocyte precursors including colony-forming units in culture (CFU-C). In tests with leukemic cells, the antibody reacted with blasts from most (8 of 9) patients with acute myelomonoblastic leukemia and from some patients with acute myeloblastic leukemia, morphologically undifferentiated acute leukemia and chronic myelogenous leukemia in blast crisis. The antibody did not react with blasts from patients with acute lymphoblastic leukemia nor with leukemic cells from patients with chronic lymphocytic leukemia.

Longitudinal analysis of quality of life in patients with undifferentiated connective tissue diseases

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Iudici M

2017-02-01

Full Text Available Michele Iudici, Rosaria Irace, Antonella Riccardi, Giovanna Cuomo, Serena Vettori, Gabriele Valentini Rheumatology Section, Department of Clinical and Experimental Medicine, Second University of Naples, Naples, Italy Introduction/objectives: To prospectively assess the quality of life (QoL of patients affected by undifferentiated connective tissue diseases (UCTDs and to identify factors associated with changes over time.Patients and methods: A total of 46 consecutive UCTD patients completed the Short-Form 36 (SF-36 questionnaire at presentation and then yearly. At each 6-month visit, all patients underwent a detailed history taking and a laboratory and physical assessment, in order to follow the evolution of the disease over time and to assess the the co-existence of fibromyalgia.Results: At presentation, scores lower than the average of the general population were detected in 34 (74% and 41 (89% patients in the physical and mental domains, respectively. No difference between patients with and without Raynaud’s phenomenon was detected. Fibromyalgia was the only independent variable associated with an impaired physical component summary score (p = 0.0009. No patient feature was found to be associated with the basal mental component summary score. During 24 months of follow-up, a significant improvement (ie, a change ≥5 from baseline in physical component summary and mental component summary scores was observed in 14 (33.3% and 20 (43.4% patients, respectively. Patients who significantly improved in the physical domain more frequently had a history of glucocorticoids intake (p < 0.001, while those who improved in the mental component more frequently had a history of either glucocorticoids (p = 0.043 or immunosuppressors (p = 0.037 intake during follow-up.Conclusion: UCTD patients perceive a worse QoL, regardless of Raynaud’s phenomenon Fibromyalgia is one of the major contributors of physical QoL, whereas no factor influencing

3D printed hyperelastic "bone" scaffolds and regional gene therapy: A novel approach to bone healing.

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Alluri, Ram; Jakus, Adam; Bougioukli, Sofia; Pannell, William; Sugiyama, Osamu; Tang, Amy; Shah, Ramille; Lieberman, Jay R

2018-04-01

The purpose of this study was to evaluate the viability of human adipose-derived stem cells (ADSCs) transduced with a lentiviral (LV) vector to overexpress bone morphogenetic protein-2 (BMP-2) loaded onto a novel 3D printed scaffold. Human ADSCs were transduced with a LV vector carrying the cDNA for BMP-2. The transduced cells were loaded onto a 3D printed Hyperelastic "Bone" (HB) scaffold. In vitro BMP-2 production was assessed using enzyme-linked immunosorbent assay analysis. The ability of ADSCs loaded on the HB scaffold to induce in vivo bone formation in a hind limb muscle pouch model was assessed in the following groups: ADSCs transduced with LV-BMP-2, LV-green fluorescent protein, ADSCs alone, and empty HB scaffolds. Bone formation was assessed using radiographs, histology and histomorphometry. Transduced ADSCs BMP-2 production on the HB scaffold at 24 hours was similar on 3D printed HB scaffolds versus control wells with transduced cells alone, and continued to increase after 1 and 2 weeks of culture. Bone formation was noted in LV-BMP-2 animals on plain radiographs at 2 and 4 weeks after implantation; no bone formation was noted in the other groups. Histology demonstrated that the LV-BMP-2 group was the only group that formed woven bone and the mean bone area/tissue area was significantly greater when compared with the other groups. 3D printed HB scaffolds are effective carriers for transduced ADSCs to promote bone repair. The combination of gene therapy and tissue engineered scaffolds is a promising multidisciplinary approach to bone repair with significant clinical potential. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1104-1110, 2018. © 2018 Wiley Periodicals, Inc.

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

DEFF Research Database (Denmark)

Jensen, Jonas; Tvedesøe, Claus; Rölfing, Jan Hendrik Duedal

2016-01-01

marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D) polycaprolactone (PCL) - hyaluronic acid - tricalcium phosphate (HT-PCL) scaffold. Population doubling (PD), alkaline phosphatase (ALP) activity, and calcium deposition were...... measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1) empty defects vs. HT-PCL scaffolds; (2) PCL scaffolds vs. HT...... a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion...

Optimization of the application of BNCT to undifferentiated thyroid cancer

International Nuclear Information System (INIS)

Dagrosa, M.A.; Thomasz, L.; Longhino, J.

2006-01-01

The possible increase in BNCT efficacy for undifferentiated thyroid carcinoma (UTC) using BPA plus BOPP and nicotinamide (NA) as a radiosensitizer on the BNCT reaction was analyzed. In these studies nude mice were transplanted with the ARO cells and after 14 days they were treated as follows: 1) Control; 2) NCT (neutrons alone); 3) NCT plus NA (100 mg/kg bw/day for 3 days); 4) BPA (350 mg/kg bw) + neutrons; 5) BPA+ NA+ neutrons; 6) BPA+BOPP (60 mg/kg bw) + neutrons. The flux of hyperthermal neutrons was 2.8 10 8 during 85 min. Neutrons alone or with NA caused some tumor growth delay, while in the BPA, BPA+NA and BPA+BOPP groups a 100% halt of tumor growth was observed. When the initial tumor volume was 50 mm 3 or less a complete cure was found in BPA+NA (2/2); BPA (1/4); BPA+BOPP (7/7). After 90 days of complete regression, recurrence of tumor was observed in 2/2 BPA/NA (2/2) and BPA+BOPP (1/7). Caspase 3 activity was increased in BPA+NA (p<0.05 vs controls). BPA plus NA increased tumor apoptosis but only the combination of BPA+BOPP increased significantly BNCT efficiency. (author)

Metabolism of monoterpenes in cell cultures of common sage (Salvia officinalis)

International Nuclear Information System (INIS)

Falk, K.L.; Gershenzon, J.; Croteau, R.

1990-01-01

Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes ( 14 C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+)-camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1,000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism

Establishment of automated culture system for murine induced pluripotent stem cells

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Koike Hiroyuki

2012-11-01

Full Text Available Abstract Background Induced pluripotent stem (iPS cells can differentiate into any cell type, which makes them an attractive resource in fields such as regenerative medicine, drug screening, or in vitro toxicology. The most important prerequisite for these industrial applications is stable supply and uniform quality of iPS cells. Variation in quality largely results from differences in handling skills between operators in laboratories. To minimize these differences, establishment of an automated iPS cell culture system is necessary. Results We developed a standardized mouse iPS cell maintenance culture, using an automated cell culture system housed in a CO2 incubator commonly used in many laboratories. The iPS cells propagated in a chamber uniquely designed for automated culture and showed specific colony morphology, as for manual culture. A cell detachment device in the system passaged iPS cells automatically by dispersing colonies to single cells. In addition, iPS cells were passaged without any change in colony morphology or expression of undifferentiated stem cell markers during the 4 weeks of automated culture. Conclusions Our results show that use of this compact, automated cell culture system facilitates stable iPS cell culture without obvious effects on iPS cell pluripotency or colony-forming ability. The feasibility of iPS cell culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

Science.gov (United States)

Freitas, Gileade P; Lopes, Helena B; Almeida, Adriana L G; Abuna, Rodrigo P F; Gimenes, Rossano; Souza, Lucas E B; Covas, Dimas T; Beloti, Marcio M; Rosa, Adalberto L

2017-09-01

One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

Kaempferol-immobilized titanium dioxide promotes formation of new bone: effects of loading methods on bone marrow stromal cell differentiation in vivo and in vitro.

Science.gov (United States)

Tsuchiya, Shuhei; Sugimoto, Keisuke; Kamio, Hisanobu; Okabe, Kazuto; Kuroda, Kensuke; Okido, Masazumi; Hibi, Hideharu

2018-01-01

Surface modification of titanium dioxide (TiO 2 ) implants promotes bone formation and shortens the osseointegration period. Kaempferol is a flavonoid that has the capacity to promote osteogenic differentiation in bone marrow stromal cells. The aim of this study was to promote bone formation around kaempferol immobilized on TiO 2 implants. There were four experimental groups. Alkali-treated TiO 2 samples (implants and discs) were used as a control and immersed in Dulbecco's phosphate-buffered saline (DPBS) (Al-Ti). For the coprecipitation sample (Al-cK), the control samples were immersed in DPBS containing 50 µg kaempferol/100% ethanol. For the adsorption sample (Al-aK), 50 µg kaempferol/100% ethanol was dropped onto control samples. The surface topography of the TiO 2 implants was observed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, and a release assay was performed. For in vitro experiments, rat bone marrow stromal cells (rBMSCs) were cultured on each of the TiO 2 samples to analyze cell proliferation, alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. For in vivo experiments, TiO 2 implants placed on rat femur bones were analyzed for bone-implant contact by histological methods. Kaempferol was detected on the surface of Al-cK and Al-aK. The results of the in vitro study showed that rBMSCs cultured on Al-cK and Al-aK promoted alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. The in vivo histological analysis revealed that Al-cK and Al-aK stimulated new bone formation around implants. TiO 2 implant-immobilized kaempferol may be an effective tool for bone regeneration around dental implants.

Scanning electron microscopic studies on bone tumors

International Nuclear Information System (INIS)

Itoh, Motoya

1978-01-01

Surface morphological observations of benign and malinant bone tumors were made by the use of scanning electron microscopy. Tumor materials were obtained directly from patients of osteogenic sarcomas, chondrosarcomas, enchondromas, giant cell tumors and Paget's sarcoma. To compare with these human tumors, the following experimental materials were also observed: P 32 -induced rat osteogenic sarcomas with their pulmonary metastatic lesions, Sr 89 -induced transplantable mouse osteogenic sarcomas and osteoid tissues arising after artificial fractures in mice. One of the most outstanding findings was a lot of granular substances seen on cell surfaces and their intercellular spaces in osteoid or chondroid forming tissues. These substances were considered to do some parts in collaborating extracellular matrix formation. Protrusions on cell surface, such as mucrovilli were more or less fashioned by these granular substances. Additional experiments revealed these substances to be soluble in sodium cloride solution. Benign osteoid forming cells, such as osteoblasts and osteoblastic osteosarcoma cells had granular substances on their surfaces and their intercellular spaces. On the other hand, undifferentiated transplantable osteosarcoma which formed on osteoid or chondroid matrix had none of these granular substances. Consequently, the difference of surface morphology between osteosarcoma cells and osteoblasts was yet to be especially concluded. (author)

Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

International Nuclear Information System (INIS)

Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

2005-01-01

Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

International Nuclear Information System (INIS)

Suzuki, Hidenori; Taguchi, Toshihiko; Tanaka, Hiroshi; Kataoka, Hideo; Li Zhenglin; Muramatsu, Keiichi; Gondo, Toshikazu; Kawai, Shinya

2004-01-01

Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

Safety assessment of bone marrow derived MSC grown in platelet-rich plasma

Directory of Open Access Journals (Sweden)

Shoji Fukuda

2015-06-01

Full Text Available The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC, which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis.

Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft.

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Pappalardo, S; Mastrangelo, F; Reale Marroccia, D; Cappello, V; Ciampoli, C; Carlino, V; Tanteri, L; Costanzo, M; Sinatra, F; Tetè, S

2008-01-01

Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.

Integrin αMβ2 is differently expressed by subsets of human osteoclast precursors and mediates adhesion of classical monocytes to bone

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Sprangers, Sara, E-mail: s.l.sprangers@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Schoenmaker, Ton, E-mail: t.schoenmaker@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Cao, Yixuan, E-mail: y.cao@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Everts, Vincent, E-mail: v.everts@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Vries, Teun J. de, E-mail: teun.devries@acta.nl [Department of Oral Cell Biology and Functional Anatomy, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands); Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam The Netherlands (Netherlands)

2017-01-01

Bone-degrading osteoclasts are formed through fusion of their monocytic precursors. In the population of human peripheral blood monocytes, three distinct subsets have been identified: classical, intermediate and non-classical monocytes. We have previously shown that when the monocyte subsets are cultured on bone, significantly more osteoclasts are formed from classical monocytes than from intermediate or non-classical monocytes. Considering that this difference does not exist when monocyte subsets are cultured on plastic, we hypothesized that classical monocytes adhere better to the bone surface compared to intermediate and non-classical monocytes. To investigate this, the different monocyte subsets were isolated from human peripheral blood and cultured on slices of human bone in the presence of the cytokine M-CSF. We found that classical monocytes adhere better to bone due to a higher expression of the integrin αMβ2 and that their ability to attach to bone is significantly decreased when the integrin is blocked. This suggests that integrin αMβ2 mediates attachment of osteoclast precursors to bone and thereby enables the formation of osteoclasts.

PCL-HA microscaffolds for in vitro modular bone tissue engineering.

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Totaro, Alessandra; Salerno, Aurelio; Imparato, Giorgia; Domingo, Concepción; Urciuolo, Francesco; Netti, Paolo Antonio

2017-06-01

The evolution of microscaffolds and bone-bioactive surfaces is a pivotal point in modular bone tissue engineering. In this study, the design and fabrication of porous polycaprolactone (PCL) microscaffolds functionalized with hydroxyapatite (HA) nanoparticles by means of a bio-safe and versatile thermally-induced phase separation process is reported. The ability of the as-prepared nanocomposite microscaffolds to support the adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in standard and osteogenic media and using dynamic seeding/culture conditions was investigated. The obtained results demonstrated that the PCL-HA nanocomposite microparticles had an enhanced interaction with hMSCs and induced their osteogenic differentiation, even without the exogenous addition of osteogenic factors. In particular, calcium deposition, alizarin red assay, histological analysis, osteogenic gene expression and collagen I secretion were assessed. The results of these tests demonstrated the formation of bone microtissue precursors after 28 days of dynamic culture. These findings suggest that PCL-HA nanocomposite microparticles represent an excellent platform for in vitro modular bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Bone marker gene expression in calvarial bones: different bone microenvironments.

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Al-Amer, Osama

2017-12-01

In calvarial mice, mesenchymal stem cells (MSCs) differentiate into osteoprogenitor cells and then differentiate into osteoblasts that differentiate into osteocytes, which become embedded within the bone matrix. In this case, the cells participating in bone formation include MSCs, osteoprogenitor cells, osteoblasts and osteocytes. The calvariae of C57BL/KaLwRijHsD mice consist of the following five bones: two frontal bones, two parietal bones and one interparietal bone. This study aimed to analyse some bone marker genes and bone related genes to determine whether these calvarial bones have different bone microenvironments. C57BL/KaLwRijHsD calvariae were carefully excised from five male mice that were 4-6 weeks of age. Frontal, parietal, and interparietal bones were dissected to determine the bone microenvironment in calvariae. Haematoxylin and eosin staining was used to determine the morphology of different calvarial bones under microscopy. TaqMan was used to analyse the relative expression of Runx2, OC, OSX, RANK, RANKL, OPG, N-cadherin, E-cadherin, FGF2 and FGFR1 genes in different parts of the calvariae. Histological analysis demonstrated different bone marrow (BM) areas between the different parts of the calvariae. The data show that parietal bones have the smallest BM area compared to frontal and interparietal bones. TaqMan data show a significant increase in the expression level of Runx2, OC, OSX, RANKL, OPG, FGF2 and FGFR1 genes in the parietal bones compared with the frontal and interparietal bones of calvariae. This study provides evidence that different calvarial bones, frontal, parietal and interparietal, contain different bone microenvironments.

Streptozotocin, Type I Diabetes Severity and Bone

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Motyl Katherine

2009-01-01

Full Text Available Abstract As many as 50% of adults with type I (T1 diabetes exhibit bone loss and are at increased risk for fractures. Therapeutic development to prevent bone loss and/or restore lost bone in T1 diabetic patients requires knowledge of the molecular mechanisms accounting for the bone pathology. Because cell culture models alone cannot fully address the systemic/metabolic complexity of T1 diabetes, animal models are critical. A variety of models exist including spontaneous and pharmacologically induced T1 diabetic rodents. In this paper, we discuss the streptozotocin (STZ-induced T1 diabetic mouse model and examine dose-dependent effects on disease severity and bone. Five daily injections of either 40 or 60 mg/kg STZ induce bone pathologies similar to spontaneously diabetic mouse and rat models and to human T1 diabetic bone pathology. Specifically, bone volume, mineral apposition rate, and osteocalcin serum and tibia messenger RNA levels are decreased. In contrast, bone marrow adiposity and aP2 expression are increased with either dose. However, high-dose STZ caused a more rapid elevation of blood glucose levels and a greater magnitude of change in body mass, fat pad mass, and bone gene expression (osteocalcin, aP2. An increase in cathepsin K and in the ratio of RANKL/OPG was noted in high-dose STZ mice, suggesting the possibility that severe diabetes could increase osteoclast activity, something not seen with lower doses. This may contribute to some of the disparity between existing studies regarding the role of osteoclasts in diabetic bone pathology. Examination of kidney and liver toxicity indicate that the high STZ dose causes some liver inflammation. In summary, the multiple low-dose STZ mouse model exhibits a similar bone phenotype to spontaneous models, has low toxicity, and serves as a useful tool for examining mechanisms of T1 diabetic bone loss.

Streptozotocin, Type I Diabetes Severity and Bone

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Motyl Katherine

2009-03-01

Full Text Available Abstract As many as 50% of adults with type I (T1 diabetes exhibit bone loss and are at increased risk for fractures. Therapeutic development to prevent bone loss and/or restore lost bone in T1 diabetic patients requires knowledge of the molecular mechanisms accounting for the bone pathology. Because cell culture models alone cannot fully address the systemic/metabolic complexity of T1 diabetes, animal models are critical. A variety of models exist including spontaneous and pharmacologically induced T1 diabetic rodents. In this paper, we discuss the streptozotocin (STZ-induced T1 diabetic mouse model and examine dose-dependent effects on disease severity and bone. Five daily injections of either 40 or 60 mg/kg STZ induce bone pathologies similar to spontaneously diabetic mouse and rat models and to human T1 diabetic bone pathology. Specifically, bone volume, mineral apposition rate, and osteocalcin serum and tibia messenger RNA levels are decreased. In contrast, bone marrow adiposity and aP2 expression are increased with either dose. However, high-dose STZ caused a more rapid elevation of blood glucose levels and a greater magnitude of change in body mass, fat pad mass, and bone gene expression (osteocalcin, aP2. An increase in cathepsin K and in the ratio of RANKL/OPG was noted in high-dose STZ mice, suggesting the possibility that severe diabetes could increase osteoclast activity, something not seen with lower doses. This may contribute to some of the disparity between existing studies regarding the role of osteoclasts in diabetic bone pathology. Examination of kidney and liver toxicity indicate that the high STZ dose causes some liver inflammation. In summary, the multiple low-dose STZ mouse model exhibits a similar bone phenotype to spontaneous models, has low toxicity, and serves as a useful tool for examining mechanisms of T1 diabetic bone loss.

Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

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Ommerborn Michelle A

2008-06-01

Full Text Available Abstract Background Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation. Methods Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor, DAG (dexamethasone, ascorbic acid and β-glycerophosphate or bone morphogenetic protein-2 (BMP-2. Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR. Results ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17. Conclusion Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.

Advances in allogenic bone graft processing and usage: preparation and evaluation of chitosan-demineralized cancellous bone powder composite scaffolds as a bone graft substitute

International Nuclear Information System (INIS)

Yongyudh Vajaradul

2008-01-01

Full text: Demineralized bone matrix (DBM) is currently used by surgeons. It usually exists as a lyophilized powder which is difficult to handle and operated. In this study, we try to improve these disadvantages by combining DBM with a biomaterial. It focuses on a natural biodegradable polymer, chitosan, to act as a temporary matrix for bone growth that easily prepare in any size and shape by using tissue engineering knowledge to get a proper temporary matrix. Thus, the development of chitosan-demineralized bone powder composite scaffold is an alternative way. Polymeric scaffold has been demonstrated to have great potential for tissue engineering because the scaffold or three dimension (3D) construct provides the necessary support for cells to proliferate, extracellular matrix deposition and vascularization of neo-tissue. Moreover, chitosan, a natural cationic polymer which its structural is similar to extracellular matrix glycosaminoblycans, is biodegradable, biocompatible, non-antigenic and biofunctional. It can enhance osteoblast cells proliferation and mineral matrix deposition in culture. The first study was to fabricate and analyze composite scaffold composed of either chitosan-demineralized cancellous bone powders or chitosan-demineralized cancellous cartilage bone powders in a ratio 50:50 and 70:30 w/w (chitosan : bone powders) based on physical properties composing of average pore diameter, mechanical integrity and swelling property. Secondly, scaffolds were evaluated in term of biological properties composing of their ability to support neo osteogenesis, including assessments of cell attachment and viability, cell morphology, and the biosynthesis of extracellular matrix. Results indicated that chitosan-demineralized cancellous bone powder composite scaffolds possessing an interconnecting, porous structure could be easily created through a simple freezing and lyophilization process. (Author)

Relationship of HS CRP and Sacroiliac Joint Inflammation in Undifferentiated Spondyloarthritis.

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Liu, Te-Jung; Chang, Cheng-Chiang; Chen, Liang-Cheng; Chu, Heng-Yi; Hsu, Chun-Sheng; Chang, Shin-Tsu

2018-01-01

Elevation of serum high sensitivity C-reactive protein (hs-CRP) level has been demonstrated as a risk factor for varying diseases, as well as a biomarker for predicting recovery after operation of lumber disc herniation. Our objective was to investigate the relationship between serum hs-CRP and sacroiliac (SI) joint inflammation in patients with undifferentiated spondyloarthritis (uSpA). In this retrospective study, we enrolled patients with uSpA who underwent hs-CRP testing between January 2007 and September 2013. Serum hs-CRP was analyzed at our central laboratory. All enrolled patients underwent skeletal scintigraphic scan with quantitative sacroiliac measurement. A total of 29 patients were enrolled with mean age 32.27 years and female:male ratio of 6:23. Pearson's correlation coefficient showed a significant difference between hs-CRP in serum and SI/S ratio in uSpA, particularly the middle part of the sacroiliac joint, either right side or left side. The significantly high concentration of serum hs-CRP might indicate a systemic inflammatory response to flare-up of the SI joint and might be an indicator of SI inflammation in uSpA.

Undifferentiated tropical febrile illness in Cordoba, Colombia: Not everything is dengue

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Salim Mattar

2017-09-01

Full Text Available Summary: In Colombia, undifferentiated tropical febrile illness (UTFI are frequent and of considerable concern. They also share many clinical features. Between 2012 and 2013 in an endemic tropical area of Cordoba, Colombia, we conducted a prospective study to establish an etiological diagnosis of UTFI. Using diagnostic tests for dengue, leptospirosis, hantavirus, malaria, rickettsia, brucellosis, hepatitis A and B on 100 patients recruited for the study. We identified 69 patients with presumed UTFI: leptospirosis (n = 27, dengue (n = 26, hantavirus infection (n = 4, malaria (n = 4, rickettsial infection (n = 2, hepatitis A (n = 1, and brucellosis (n = 1; no hepatitis B cases were detected. Co-infections with malaria and leptospirosis (n = 1, hepatitis A and dengue (n = 1, hantavirus and dengue (n = 1, hantavirus, dengue, and leptospirosis (n = 1 were also identified. No etiologic agent was identified for 31 patients. We conclude that other etiologic agents besides dengue virus deserve greater attention by physicians and public health authorities in tropical area of Colombia. Keywords: Leptospirosis, Hantaviruses, Malaria, Vector-borne diseases, Zoonotic diseases

Relationship of HS CRP and Sacroiliac Joint Inflammation in Undifferentiated Spondyloarthritis

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Liu, Te-Jung; Chang, Cheng-Chiang; Chen, Liang-Cheng; Chu, Heng-Yi; Hsu, Chun-Sheng; Chang, Shin-Tsu

2018-01-01

Abstract Objective Elevation of serum high sensitivity C-reactive protein (hs-CRP) level has been demonstrated as a risk factor for varying diseases, as well as a biomarker for predicting recovery after operation of lumber disc herniation. Our objective was to investigate the relationship between serum hs-CRP and sacroiliac (SI) joint inflammation in patients with undifferentiated spondyloarthritis (uSpA). Methods In this retrospective study, we enrolled patients with uSpA who underwent hs-CRP testing between January 2007 and September 2013. Serum hs-CRP was analyzed at our central laboratory. All enrolled patients underwent skeletal scintigraphic scan with quantitative sacroiliac measurement. Results A total of 29 patients were enrolled with mean age 32.27 years and female:male ratio of 6:23. Pearson’s correlation coefficient showed a significant difference between hs-CRP in serum and SI/S ratio in uSpA, particularly the middle part of the sacroiliac joint, either right side or left side. The significantly high concentration of serum hs-CRP might indicate a systemic inflammatory response to flare-up of the SI joint and might be an indicator of SI inflammation in uSpA. PMID:29785410

The effect of Rayleigh-Taylor instabilities on the thickness of undifferentiated crust on Kuiper Belt Objects

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Rubin, Mark E.; Desch, Steven J.; Neveu, Marc

2014-07-01

Previous calculations of the internal structure and thermal evolution of Kuiper Belt Objects (KBOs) by Desch et al. (Desch, S.J., Cook, J.C., Doggett, T.C., Porter, S.B. [2009]. Icarus 202, 694-714) have predicted that KBOs should only partially differentiate, with rock and ice separating into a rocky core and icy mantle, below an undifferentiated crust of ice and rock. This crust is thermally insulating and enhances the ability of subsurface liquid to persist within KBOs. A dense rock/ice layer resting on an icy mantle is gravitationally unstable and prone to Rayleigh-Taylor (RT) instabilities, and may potentially overturn. Here we calculate the ability of RT instabilities to act in KBOs, and determine the thickness of undifferentiated crusts. We have used previously calculated growth rates of the RT instability to determine the critical viscosity of ice needed for the RT instability to operate. We calculate the viscosity of ice at the cold temperatures and long timescales relevant to KBOs. We find that crustal overturn is only possible where the temperature exceeds about 150 K, and that RT instabilities cannot act on geological timescales within about 60 km of the surfaces of a KBO like Charon. Although this crustal thickness is less than the 85 km previously calculated by Desch et al. (Desch, S.J., Cook, J.C., Doggett, T.C., Porter, S.B. [2009]. Icarus 202, 694-714), it is still significant, representing ≈25% of the mass of the KBO. We conclude that while RT instabilities may act in KBOs, they do not completely overturn their crusts. We calculate that Saturn’s moon Rhea should only partially differentiate, resulting in a moment of inertia C/MR2≈0.38.

The effect of magnesium ion implantation into alumina upon the adhesion of human bone derived cells

International Nuclear Information System (INIS)

Howlett, C.R.; Zreiqat, H.; O'Dell, R.; Noorman, J.; Evans, P.; Dalton, B.A.; McFarland, C.; Steele, J.G.

1994-01-01

Our group is investigating the potential of modifying the surface atomic layers of biomaterials by ion beam implantation in order to stimulate adhesion of bone cells to these treated biomaterials. In this study alumina that had been implanted with magnesium ions (Mg)-(Al 2 O 3 ), was compared to unmodified alumina (Al 2 O 3 ) for the adhesion of cells cultured from explanted human bone. The attachment and spreading of cultured human bone derived cells onto (Mg)-(Al 2 O 3 ) was significantly enhanced as compared to Al 2 O 3 . The role of adsorption of serum adhesive glycoproteins firbronectin (Fn) and vitronectin (Vn) in the adhesion of human bone derived cells to (Mg)-(Al 2 O 3 ) was determined. (Author)

Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

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Chengbo Tan

2016-01-01

Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

Transgenic Mouse Model for Reducing Oxidative Damage in Bone

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Schreurs, A.-S.; Torres, S.; Truong, T.; Kumar, A.; Alwood, J. S.; Limoli, C. L.; Globus, R. K.

2014-01-01

Exposure to musculoskeletal disuse and radiation result in bone loss; we hypothesized that these catabolic treatments cause excess reactive oxygen species (ROS), and thereby alter the tight balance between bone resorption by osteoclasts and bone formation by osteoblasts, culminating in bone loss. To test this, we used transgenic mice which over-express the human gene for catalase, targeted to mitochondria (MCAT). Catalase is an anti-oxidant that converts the ROS hydrogen peroxide into water and oxygen. MCAT mice were shown previously to display reduced mitochondrial oxidative stress and radiosensitivity of the CNS compared to wild type controls (WT). As expected, MCAT mice expressed the transgene in skeletal tissue, and in marrow-derived osteoblasts and osteoclast precursors cultured ex vivo, and also showed greater catalase activity compared to wildtype (WT) mice (3-6 fold). Colony expansion in marrow cells cultured under osteoblastogenic conditions was 2-fold greater in the MCAT mice compared to WT mice, while the extent of mineralization was unaffected. MCAT mice had slightly longer tibiae than WT mice (2%, P less than 0.01), although cortical bone area was slightly lower in MCAT mice than WT mice (10%, p=0.09). To challenge the skeletal system, mice were treated by exposure to combined disuse (2 wk Hindlimb Unloading) and total body irradiation Cs(137) (2 Gy, 0.8 Gy/min), then bone parameters were analyzed by 2-factor ANOVA to detect possible interaction effects. Treatment caused a 2-fold increase (p=0.015) in malondialdehyde levels of bone tissue (ELISA) in WT mice, but had no effect in MCAT mice. These findings indicate that the transgene conferred protection from oxidative damage caused by treatment. Unexpected differences between WT and MCAT mice emerged in skeletal responses to treatment.. In WT mice, treatment did not alter osteoblastogenesis, cortical bone area, moment of inertia, or bone perimeter, whereas in MCAT mice, treatment increased these

Specific receptors for epidermal growth factor in human bone tumour cells and its effect on synthesis of prostaglandin E2 by cultured osteosarcoma cell line

International Nuclear Information System (INIS)

Hirata, Y.; Uchihashi, M.; Nakashima, H.; Fujita, T.; Matsukura, S.; Matsui, K.

1984-01-01

Using tumour cell lines derived from human bone tumours, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator in many tissues, and its effect on synthesis of prostaglandin (PG) E 2 , a potent bone-resorbing factor, by cultured osteosarcoma cell line were studied. Three tumour cell lines, one osteosarcoma (HOSO) and two giant cell tumours of the bone (G-1 and G-2), all possessed specific binding sites for 125 I-labelled EGF: the apparent dissociation constant was approximately 4-10 x 10 -10 M and the maximal binding capacity was 50 000-80 000 sites/cell. EGF had no mitogenic effect in these cell lines. However, these cell lines did not have specific binding sites for 125 I-labelled parathyroid hormone (PTH) or calcitonin. HOSO line produced and secreted PGE 2 into medium, while no significant amount of PGE 2 was demonstrated in G-1 or G-2 line. EGF significantly stimulated PGE 2 production in HOSO line in a dose-dependent manner (0.5-50 ng/ml); its stimulatory effect was completely abolished by indomethacin, an inhibitor of PG biosynthesis. Exogenous PGE 1 significantly stimulated cyclic AMP formation in HOSO line, whereas PGFsub(2α) PTH, calcitonin, or EGF had no effect. None of these calcium-regulating hormones affected cyclic AMP generation in either G-1 of G-2 line. These data indicate that human bone tumour cells have specific EGF receptors unrelated to cell growth, and suggest that EGF may be involved in bone resorption through a PGE 2 -mediated process in human osseous tissues. (author)

Modeling evolution of the mind and cultures: emotional Sapir-Whorf hypothesis

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Perlovsky, Leonid I.

2009-05-01

Evolution of cultures is ultimately determined by mechanisms of the human mind. The paper discusses the mechanisms of evolution of language from primordial undifferentiated animal cries to contemporary conceptual contents. In parallel with differentiation of conceptual contents, the conceptual contents were differentiated from emotional contents of languages. The paper suggests the neural brain mechanisms involved in these processes. Experimental evidence and theoretical arguments are discussed, including mathematical approaches to cognition and language: modeling fields theory, the knowledge instinct, and the dual model connecting language and cognition. Mathematical results are related to cognitive science, linguistics, and psychology. The paper gives an initial mathematical formulation and mean-field equations for the hierarchical dynamics of both the human mind and culture. In the mind heterarchy operation of the knowledge instinct manifests through mechanisms of differentiation and synthesis. The emotional contents of language are related to language grammar. The conclusion is an emotional version of Sapir-Whorf hypothesis. Cultural advantages of "conceptual" pragmatic cultures, in which emotionality of language is diminished and differentiation overtakes synthesis resulting in fast evolution at the price of self doubts and internal crises are compared to those of traditional cultures where differentiation lags behind synthesis, resulting in cultural stability at the price of stagnation. Multi-language, multi-ethnic society might combine the benefits of stability and fast differentiation. Unsolved problems and future theoretical and experimental directions are discussed.

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

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Babur, Betul Kul; Futrega, Kathryn; Lott, William B; Klein, Travis Jacob; Cooper-White, Justin; Doran, Michael Robert

2015-09-01

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.

The r.b.e. of different-energy neutrons as determined by human bone-marrow cell-culture techniques

International Nuclear Information System (INIS)

Boeyum, A.; Carsten, A.L.; Chikkappa, G.; Cook, L.; Bullis, J.; Honikel, L.; Cronkite, E.P.

1978-01-01

The effect of X-rays and different-energy neutrons on human bone-marrow cells was studied using two different cell-culture techniques - diffusion chamber (DC) growth and colony formation in vitro (CFU-C). Based on the survival and proliferative granulocytes in DC on day 13, the D 0 value was 80 rad with X-rays, and 117 rad as measured by the CFU-C assay. The D 0 values for neutrons depended on the radiation source and the energy level. The r.b.e. values, which dropped with increasing energy levels of mono-energetic neutrons, were (i) 0.44 MeV; DC 3.7, CFU-C 4.1; (ii) 6 MeV; DC 1.8, CFU-C 2.0; (iii) 15 MeV; DC 1.6, CFU-C 1.6; (iv) fission neutrons; DC 2.6, CFU-C 2.4. (author)

Evaluation of low doses of gamma irradiation in the formation of mineralization nodules in osteoblasts culture

Energy Technology Data Exchange (ETDEWEB)

Targino, Bárbara; Pinto, Thais Lazarine; Silva, Evily Fernandes; Somessari, E.S.R.; Bellini, Maria Helena; Affonso, Regina [Instituto De Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil)

2017-07-01

Full text: Introduction: Osteoblasts are specialized fibroblasts that secrete and mineralize the bone matrix. The mineralized extracellular matrix is mainly composed of type I collagen, osteocalcin, and the inorganic mineral hydroxylapatite1. The use of radiation as therapy in some cancers causes great bone loss. However, low dose radiation may have the opposite effect. Low dose X-irradiation on osteoblastic culture had effects on proliferation and differentiation with increase of mineralization nodules2. However, there is little information on the potential therapeutic efficacy of low-dose gamma-irradiation in the formation of mineralization nodules. Objective: To evaluate the effects of irradiation with 60Co γ-rays in low doses in the formation of mineralization nodules in culture of osteoblasts. Methods: MC3T3-E1 cells were bought by the Banco de Células do Rio de Janeiro, Brazil (MC3T3-E1 Subclone 14). The cells were cultured in α-MEM medium consisting of 10% FBS and without β-glycerophosphate and L-ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001) (Zhao Y, Guan H, Liu S et al. Biol. Pharm. Bull. 2005, 28(8):1371-1376). Plating efficiency assays: cells were plated at a density of 100 cell/plate into 60 mm Petri dishes. After 14 days the places were stained with violet crystal and the colonies were counted. -glycerophosphate and 50 mg/ml ascorbic acid, and analyzed on days 7, 14 and 21. Osteoblast culture irradiation assay: cells were plated at a density of 1 x 105 cells/plate on 60 mm dishes and the next day were irradiated by 60Co source with 0 (as the control), 0.5, 1.0, 1.5 and 2.0 Gy using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commission Ltd. (CTR-IPEN). On day 21 of culture, undifferentiated (without ascorbic acid and β-glycerophosphate), differentiating cells (0 Gy) and irradiated cells at different doses, the medium was removed, cells were washed with phosphate buffer saline, fixed with 70% ethyl alcohol and

Evaluation of low doses of gamma irradiation in the formation of mineralization nodules in osteoblasts culture

International Nuclear Information System (INIS)

Targino, Bárbara; Pinto, Thais Lazarine; Silva, Evily Fernandes; Somessari, E.S.R.; Bellini, Maria Helena; Affonso, Regina

2017-01-01

Full text: Introduction: Osteoblasts are specialized fibroblasts that secrete and mineralize the bone matrix. The mineralized extracellular matrix is mainly composed of type I collagen, osteocalcin, and the inorganic mineral hydroxylapatite1. The use of radiation as therapy in some cancers causes great bone loss. However, low dose radiation may have the opposite effect. Low dose X-irradiation on osteoblastic culture had effects on proliferation and differentiation with increase of mineralization nodules2. However, there is little information on the potential therapeutic efficacy of low-dose gamma-irradiation in the formation of mineralization nodules. Objective: To evaluate the effects of irradiation with 60Co γ-rays in low doses in the formation of mineralization nodules in culture of osteoblasts. Methods: MC3T3-E1 cells were bought by the Banco de Células do Rio de Janeiro, Brazil (MC3T3-E1 Subclone 14). The cells were cultured in α-MEM medium consisting of 10% FBS and without β-glycerophosphate and L-ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001) (Zhao Y, Guan H, Liu S et al. Biol. Pharm. Bull. 2005, 28(8):1371-1376). Plating efficiency assays: cells were plated at a density of 100 cell/plate into 60 mm Petri dishes. After 14 days the places were stained with violet crystal and the colonies were counted. -glycerophosphate and 50 mg/ml ascorbic acid, and analyzed on days 7, 14 and 21. Osteoblast culture irradiation assay: cells were plated at a density of 1 x 105 cells/plate on 60 mm dishes and the next day were irradiated by 60Co source with 0 (as the control), 0.5, 1.0, 1.5 and 2.0 Gy using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commission Ltd. (CTR-IPEN). On day 21 of culture, undifferentiated (without ascorbic acid and β-glycerophosphate), differentiating cells (0 Gy) and irradiated cells at different doses, the medium was removed, cells were washed with phosphate buffer saline, fixed with 70% ethyl alcohol and

Design and optimization of a tissue-engineered bone graft substitute

Science.gov (United States)

Shimko, Daniel Andrew

2004-12-01

In 2000, 3.1 million surgical procedures on the musculoskeletal system were reported in the United States. For many of these cases, bone grafting was essential for successful fracture stabilization. Current techniques use intact bone obtained either from the patient (autograft) or a cadaver (allograft) to repair large defects, however, neither source is optimal. Allografts suffer integration problems, and for autografts, the tissue supply is limited. Because of these shortcomings, and the high demand for graft tissues, alternatives are being explored. To successfully engineer a bone graft replacement, one must employ a three pronged research approach, addressing (1) the cells that will inhabit the new tissue, (2) the culture environment that these cells will be exposed to, and (3) the scaffold in which these cells will reside. The work herein examines each of these three aspects in great detail. Both adult and embryonic stem cells (ESCs) were considered for the tissue-engineered bone graft. Both exhibited desirable qualities, however, neither were optimal in all categories examined. In the end, the possibility of teratoma formation and ethical issues surrounding ESCs, made the use of adult marrow-derived stem cells in the remaining experiments obligatory. In subsequent experiments, the adult stem cells' ability to form bone was optimized. Basic fibroblast growth factor, fetal bovine serum, and extracellular calcium supplementation studies were all performed. Ultimately, adult stem cells cultured in alpha-MEM supplemented with 10% fetal bovine serum, 10mM beta-glycerophosphate, 10nM dexamethasone, 50mug/ml ascorbic acid, 1%(v/v) antibiotic/antimycotic, and 10.4mM CaCl2 performed the best, producing nearly four times more mineral than any other medium formulation. Several scaffolds were then investigated including those fabricated from poly(alpha-hydroxy esters), tantalum, and poly-methylmethacrylate. In the final study, the most appealing cell type, medium

Pullulan microcarriers for bone tissue regeneration

Energy Technology Data Exchange (ETDEWEB)

Aydogdu, Hazal [Middle East Technical University, Department of Biomedical Engineering, Ankara 06800 (Turkey); Keskin, Dilek [Middle East Technical University, Department of Biomedical Engineering, Ankara 06800 (Turkey); Middle East Technical University, Department of Engineering Sciences, Ankara 06800 (Turkey); METU BIOMATEN Center of Excellence in Biomaterials and Tissue Engineering, Ankara 06800 (Turkey); Baran, Erkan Turker, E-mail: erkanturkerbaran@gmail.com [METU BIOMATEN Center of Excellence in Biomaterials and Tissue Engineering, Ankara 06800 (Turkey); Tezcaner, Aysen, E-mail: tezcaner@metu.edu.tr [Middle East Technical University, Department of Biomedical Engineering, Ankara 06800 (Turkey); Middle East Technical University, Department of Engineering Sciences, Ankara 06800 (Turkey); METU BIOMATEN Center of Excellence in Biomaterials and Tissue Engineering, Ankara 06800 (Turkey)

2016-06-01

Microcarrier systems offer a convenient way to repair bone defects as injectable cell carriers that can be applied with small incisions owing to their small size and spherical shape. In this study, pullulan (PULL) microspheres were fabricated and characterized as cell carriers for bone tissue engineering applications. PULL was cross-linked by trisodium trimetaphosphate (STMP) to enhance the stability of the microspheres. Improved cytocompatibility was achieved by silk fibroin (SF) coating and biomimetic mineralization on the surface by incubating in simulated body fluid (SBF). X-ray diffraction (XRD), scanning electron microscopy (SEM) and fluorescent microscopy analysis confirmed biomimetic mineralization and SF coating on microspheres. The degradation analysis revealed that PULL microspheres had a slow degradation rate with 8% degradation in two weeks period indicating that the microspheres would support the formation of new bone tissue. Furthermore, the mechanical tests showed that the microspheres had a high mechanical stability that was significantly enhanced with the biomimetic mineralization. In vitro cell culture studies with SaOs-2 cells showed that cell viability was higher on SF and SBF coated microspheres on 7th day compared to PULL ones under dynamic conditions. Alkaline phosphatase activity was higher for SF coated microspheres in comparison to uncoated microspheres when dynamic culture condition was applied. The results suggest that both organic and inorganic surface modifications can be applied on PULL microspheres to prepare a biocompatible microcarrier system with suitable properties for bone tissue engineering. - Highlights: • Porous PULL microspheres were prepared as cell carrier for the first time. • Mineralization on the microspheres improved their mechanical properties. • Mineralization and SF coating enhanced cell proliferation on PULL microspheres.

Pullulan microcarriers for bone tissue regeneration

International Nuclear Information System (INIS)

Aydogdu, Hazal; Keskin, Dilek; Baran, Erkan Turker; Tezcaner, Aysen

2016-01-01

Microcarrier systems offer a convenient way to repair bone defects as injectable cell carriers that can be applied with small incisions owing to their small size and spherical shape. In this study, pullulan (PULL) microspheres were fabricated and characterized as cell carriers for bone tissue engineering applications. PULL was cross-linked by trisodium trimetaphosphate (STMP) to enhance the stability of the microspheres. Improved cytocompatibility was achieved by silk fibroin (SF) coating and biomimetic mineralization on the surface by incubating in simulated body fluid (SBF). X-ray diffraction (XRD), scanning electron microscopy (SEM) and fluorescent microscopy analysis confirmed biomimetic mineralization and SF coating on microspheres. The degradation analysis revealed that PULL microspheres had a slow degradation rate with 8% degradation in two weeks period indicating that the microspheres would support the formation of new bone tissue. Furthermore, the mechanical tests showed that the microspheres had a high mechanical stability that was significantly enhanced with the biomimetic mineralization. In vitro cell culture studies with SaOs-2 cells showed that cell viability was higher on SF and SBF coated microspheres on 7th day compared to PULL ones under dynamic conditions. Alkaline phosphatase activity was higher for SF coated microspheres in comparison to uncoated microspheres when dynamic culture condition was applied. The results suggest that both organic and inorganic surface modifications can be applied on PULL microspheres to prepare a biocompatible microcarrier system with suitable properties for bone tissue engineering. - Highlights: • Porous PULL microspheres were prepared as cell carrier for the first time. • Mineralization on the microspheres improved their mechanical properties. • Mineralization and SF coating enhanced cell proliferation on PULL microspheres.

Comparative study of two boron compounds (BPA and BOPP) for the application of BNCT to an animal model of undifferentiated thyroid cancer

International Nuclear Information System (INIS)

Dagrosa, Maria A.; Viaggi, Mabel; Juvenal, Guillermo; Pisarev, Mario A.

2003-01-01

Boron neutron capture therapy (BNCT) is based on the selective uptake of certain boron compounds by tumors. Once the uptake, relative to normal tissues, is equal of greater than 3, the tumoral area is irradiated with an appropriate neutron beam. The 10 B is then converted into 11 B and this decays releasing an atom of Li, gamma rays and alpha particles. These latter have a high linear energy transfer (LET) and will cause local damage, eventually killing the tumoral cells. At the present time several clinical trials are being conducted in different countries to treat patients with glioblastoma multiform and melanomas. So far the results obtained, specially with this last disease, are quite encouraging. Undifferentiated thyroid cancer (UTC) is a very aggressive tumor which does not respond to the therapies available at the present. Usually it has a very bad prognosis with a very short survival period. We have previously shown that the human UTC cell line ARO has an uptake of borophenylanine (BPA) significantly greater than normal thyroid or than human follicular adenoma cells in culture. Moreover, an animal model for UTC was developed in our laboratory by transplanting the human ARO cells into nude mice. This model closely resembles the evolution of human disease and even produces lung metastasis, like the human. In the present studies we have compared the uptake of two boron compounds: BPA and boronated porphyrin (BOPP). BPA was administered via ip in a dose of 600 mg/kg body weight, while BOPP was given either ip or iv, in doses of 10 and 100 mg/kg body weight. The animals were sacrificed at different times after the injection: up to 150 min for BPA and after 24 h with BOPP. The concentration of boron was determined by ICP-AES. The results obtained showed that the uptake of BPA was significantly greater in the tumoral area and in the infiltrated surrounding skin than in the other organs examined (liver, kidney, lung, mice thyroid, blood, spleen and distal skin

Successful disinfection of femoral head bone graft using high hydrostatic pressure.

Science.gov (United States)

van de Sande, Michiel A J; Bovée, Judith V M G; van Domselaar, Mark; van Wijk, Marja J; Sanders, Ingrid; Kuijper, Ed

2017-12-20

The current standard for sterilization of potentially infected bone graft by gamma irradiation and thermal or chemical inactivation potentially deteriorates the biomechanical properties of the graft. We performed an in vitro experiment to evaluate the use of high hydrostatic pressure (HHP); which is widely used as a disinfection process in the food processing industry, to sterilize bone grafts. Four femoral heads were divided into five parts each, of which 16 were contaminated (in duplicate) with 10 5 -10 7  CFU/ml of Staphylococcus epidermidis, Bacillus cereus, or Pseudomonas aeruginosa or Candida albicans, respectively. Of each duplicate, one sample was untreated and stored similarly as the treated sample. The remaining four parts were included as sterile control and non-infected control. The 16 parts underwent HHP at the high-pressure value of 600 MPa. After HHP, serial dilutions were made and cultured on selective media and into enrichment media to recover low amounts of microorganism and spores. Three additional complete femoral heads were treated with 0, 300 and 600 MPa HHP respectively for histological evaluation. None of the negative-control bone fragments contained microorganisms. The measured colony counts in the positive-control samples correlated excellent with the expected colony count. None of the HHP treated bone fragments grew on culture plates or enrichment media. Histological examination of three untreated femoral heads showed that the bone structure remained unchanged after HHP. Sterilizing bone grafts by high hydrostatic pressure was successful and is a promising technique with the possible advantage of retaining biomechanical properties of bone tissue.

False Positive Radioiodinated Metaiodobenzylguanidine (123I-MIBG Uptake in Undifferentiated Adrenal Malignant Tumor

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Hee Soo Jung

2015-01-01

Full Text Available 123I-Metaiodobenzylguanidine (123I-MIBG scintigraphy is a widely used functional imaging tool with a high degree of sensitivity and specificity in diagnosis of pheochromocytoma. However, rare cases of false positive reactions have been reported. A 67-year-old male patient was admitted with epigastric pain. Abdominal computed tomography (CT revealed a heterogeneous left adrenal mass 6 cm in diameter; following hormone testing, 123I-MIBG scintigraphy was performed to determine the presence of pheochromocytoma, which confirmed eccentric uptake by a large left adrenal gland mass. Chest CT and PET-CT confirmed metastatic lymphadenopathy; therefore, endobronchial ultrasound transbronchial needle aspiration was performed. Metastatic carcinoma of unknown origin was suspected from a lymph node biopsy, and surgical resection was performed for definitive diagnosis and correction of excess hormonal secretion. A final diagnosis of undifferentiated adrenal malignant tumor was rendered, instead of histologically malignant pheochromocytoma, despite the uptake of 123I-MIBG demonstrated by scintigraphy.

Growth of intestinal epithelium in organ culture is dependent on EGF signalling

International Nuclear Information System (INIS)

Abud, Helen E.; Watson, Nadine; Heath, Joan K.

2005-01-01

Differentiation of endoderm into intestinal epithelium is initiated at E13.5 of mouse development when there are significant changes in morphology resulting in the conversion of undifferentiated stratified epithelium into a mature epithelial monolayer. Here we demonstrate that monolayer formation is associated with the selective apoptosis of superficial cells lining the lumen while cell proliferation is progressively restricted to cells adjacent to the basement membrane. We describe an innovative embryonic gut culture system that maintains the three-dimensional architecture of gut and in which these processes are recapitulated in vitro. Explants taken from specific regions of the gut and placed into organ culture develop and express molecular markers (Cdx1, Cdx2 and A33 antigen) in the same spatial and temporal pattern observed in vivo indicating that regional specification is maintained. Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase using the specific inhibitor AG1478 significantly reduced the proliferation and survival of cells within the epithelial cell layer of cultured gut explants. This demonstrates an essential role for the EGF signalling pathway during the early stages of intestinal development

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting.

Science.gov (United States)

Wong, Chui-Wei; Chen, You-Tzung; Chien, Chung-Liang; Yu, Tien-Yu; Rwei, Syang-Peng; Hsu, Shan-Hui

2018-01-01

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigation of in vitro bone cell adhesion and proliferation on Ti using direct current stimulation

International Nuclear Information System (INIS)

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

2012-01-01

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

The Bisphosphonate Clodronate Modifying Hydroxyapatite Bioceramics for Bone Scaffold

Institute of Scientific and Technical Information of China (English)

无

2005-01-01

To investigate the efficiency of clodronate modifying HA bioceramics , and to evaluate the effect of clodronate modifying HA bioceramies on the cells in vitro, clodronate modified the porous HA bioceramics for bone scaffold by chelation. The outermost layer of the specimens was analyzed by XPS and FI- IR. The depth profile was investigated by the argon-ion sputtering method. The cell culture test was conducted using MC3 T3-E1 osteoblastic cells. The cells were inoculated and cultured on the scaffolds. Morphological observation of the cells,MTT test and ALP activity test evaluated the cell attachment, proliferation and activity on the materials. Characteristic peaks in XPS and FT-IR spectra indicated clodronate being immobilized on the surface of the bioceramics.The cell culture test in cell quantity and morphology indicated active proliferation of the cells on the scaffolds. The ALP activity of the cells cultured for 3d and 7 d on clodronate- HA bioceramics was slightly higher than that on HA bioceramics, but the difference was not significant. This result indicated that clodronate- HA bioceramics had favorable cytocompatibility to be used as bone scaffold with potential ability to improve osteogenesis.

Bacterial infections associated with allogenic bone transplantation

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Stepanović Željko Lj.

2015-01-01

Full Text Available Background/Aim. Bone allografts are frequently used in orthopedic reconstructive procedures carrying a high risk for recipients. To assess the nature and frequency of allograft contamination and associated surgical infection the case records from our institutional bone bank were reviewed. Methods. We retrospectively analyzed the microbiology of discarded bone allografts and the surgical site of the recipients. A case series of patients who acquired surgical site infection after allogenic bone transplantation was presented. Swab culturing was conducted on 309 femoral heads from living donors who underwent partial and total hip arthroplasty between January 2007 and December 2013. To prevent potential bone allograft contamination we used saline solution of 2.0 mg/ml of amikacin during thawing. The overall infection rate was analyzed in 197 recipients. Results. Of the 309 donated femoral heads, 37 were discarded due to bacterial contamination, giving the overall contamination rate of 11.97%. The postoperative survey of 213 bone allotransplantations among 197 recipients showed the infection rate of 2.03%. The coagulase-negative Staphylococcus was the most commonly identified contaminant of bone allografts and recipient surgical sites. Conclusion. The allograft contamination rate and the infection rate among recipients in our institution are in accordance with the international standards. The coagulase-negative Staphylococcus was the most commonly identified contaminant of bone allografts and recipient surgical sites. There is no strong evidence that surgical site infections were associated with bone allograft utilization. We plan further improvements in allograft handling and decontamination with highly concentrated antibiotic solutions in order to reduce infection risk for recipients.

Undifferentiated seronegative spondyloarthritis with inflammatory bowel disease and a family history of psoriasis. Sicca syndrome

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Norma Marigliano

2013-04-01

Full Text Available Background: Seronegative spondyloarthritis is characterized by the presence of subcutaneous nodules, asymmetrical peripheral arthritis, sacroileitis with or without spondylitis, and rheumatoid-factor negativity. Other common clinical manifestations include oral ulcers, conjunctivitis, and cutaneous lesions such as psoriasis. Familial aggregation has also been described. According to the 1986 classification, corresponding clinical entities include ankylosing spondylitis, psoriatic arthritis, Reiter’s syndrome, arthritis associated with inflammatory bowel disease (IBD, and undifferentiated spondyloarthritis. The disease is also frequently associated with the HLA B27 antigen. From the clinical point of view, there are often incomplete forms of spondyloarthritis, such as reactive arthritis triggered by asymptomatic infections, psoriatic arthritis without psoriasis itself, initial phases of specific forms of spondyloarthritis or the phase of ankylosing spondylitis characterized by sacroiliac lesions, and all forms that remain undifferentiated for long periods of time. Moreover, there are close relations between arthropathy and IBDs, such as Crohn’s disease, ulcerative colitis, and Whipple’s syndrome. Recently, microscopic inflammatory bowel lesions and psoriatic arthritis have been described. Case report: A 30-year-old man (HLA B27-negative who had been vaccinated against TBC and HBV presented with a 6-year history of recurrent episodes of predominantly left-sided sciatica. The pain was worse at night and during rest. He was suffering from bilateral sacroileitis without spondylitis. Three to five times a day, usually after eating, he passed watery feces containing mucous and small amounts of bright red blood. Colonoscopy revealed pancolitis with histological evidence of chronic inflammation interspersed with areas of acute inflammation, edema, hyperemia, and glandular distortion. One year later, the clinical manifestations and histological

Expression of fra(10)(q25) in peripheral blood and bone marrow in familial neutropenia.

OpenAIRE

Holmes, J A; Thompson, P W

1988-01-01

We report on the expression of fra(10)(q25) in a mother and daughter with familial chronic neutropenia. Differences in expression of the fragile site in bone marrow cells compared with peripheral blood lymphocytes were observed. Short term bone marrow cultures in complete medium showed high levels of spontaneous expression, whereas in 72 hour stimulated blood cultures it was virtually absent. There were also differences in the types of lesions found; chromosome type lesions predominated in th...

Characterization of human erythroid burst-promoting activity derived from bone marrow conditioned media

International Nuclear Information System (INIS)

Porter, P.N.; Ogawa, M.

1982-01-01

Bone marrow conditioned media (BMCM) increases burst number and the incorporation of 59 Fe into heme by bursts when peripheral blood or bone marrow cells are cultured at limiting serum concentrations. Burst-promoting activity (BPA) has now been purified approximately 300-fold from this source by ion-exchange chromatography on DEAE-Sephadex and absorption chromatography on hydroxyapatite agarose gel. Marrow BPA increased burst number and hemoglobin (Hb) synthesis in a dose-dependent manner. A larger increase in Hb synthesis than in burst number was consistently observed, which was probably a consequence of the increase in the number of cells per burst that occurs in the presence of BPA. The role of BPA in culture could be distinguished from erythropoietin (Ep), since no bursts grew in the absence of Ep, whether or not BPA was present, and since it had no effect on the growth of erythroid colonies scored at day 5 of culture. Our purified fraction did not support the growth of CFU-C in culture. Activity was stable at temperatures of 70 degrees C or lower for 10 min; exposure to 80 degrees C resulted in approximately 50% loss of activity. BPA was completely inactivated by treatment at 100 degrees C for 10 min. Thus, human bone marrow cells produce a heat-sensitive factor that specifically promotes the growth of early erythroid progenitors in culture

Calcium Concentration in Culture Medium as a Nondestructive and Rapid Marker of Osteogenesis.

Science.gov (United States)

Tanikake, Yohei; Akahane, Manabu; Furukawa, Akira; Tohma, Yasuaki; Inagaki, Yusuke; Kira, Tsutomu; Tanaka, Yasuhito

2017-06-09

Artificial bones made of β-tricalcium phosphate (β-TCP) combined with bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used for effective reconstruction of bone defects caused by genetic defects, traumatic injury, or surgical resection of bone tumors. However, the selection of constructs with high osteogenic potential before implantation is challenging. The purpose of this study was to determine whether the calcium concentration in BM-MSC culture medium can be used as a nondestructive and simple osteogenic marker for selecting tissue-engineered grafts constructed using β-TCP and BM-MSCs. We prepared three cell passages of BM-MSCs derived from three 7-week-old, male Fischer 344 rats; the cells were cultured in osteoinductive medium in the presence of β-TCP for 15 days. The medium was replaced with fresh medium on day 1 in culture and subsequently changed every 48 h; it was collected for measurement of osteocalcin secretion and calcium concentration by enzyme-linked immunosorbent assay and X-ray fluorescence spectrometry, respectively. After cultivation, the constructs were implanted subcutaneously into the backs of recipient rats. Four weeks after implantation, the alkaline phosphatase (ALP) activity and osteocalcin content of the constructs were measured. A strong inverse correlation was observed between the calcium concentration in the medium and the ALP activity and osteocalcin content of the constructs, with Pearson's correlation coefficients of 0.92 and 0.90, respectively. These results indicate that tissue-engineered bone with high osteogenic ability can be selected before implantation based on low calcium content of the culture medium, resulting in successful bone formation after implantation. This nondestructive, simple method shows great promise for assessing the osteogenic ability of tissue-engineered bone.

Lethal graft-versus-host disease: modification with allogeneic cultured donor cells

International Nuclear Information System (INIS)

Mauch, P.; Lipton, J.M.; Hamilton, B.; Obbagy, J.; Kudisch, M.; Nathan, D.; Hellman, S.

1984-01-01

The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta-positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90-day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences

[Donor age affects on the «behavior» and the sensibility bone marrow cells in on copper ion of the primary culture].

Science.gov (United States)

Bozhkov, A I; Ohiienko, S L; Kuznetsova, Yu A; Bondar', A Yu; Marchenko, V P; Gumennaya, M S

2017-01-01

The changes of bone marrow cells (BMC) number in the primary culture from 0 to 96 hours, the pattern (the distribution of cells) of cells morphotypes and «lifespan» (the time of cell life after isolation) of myelocytes, metamyelocytes, band and segmented neutrophils, isolated of the young (3 months) and old (20months) animals, were investigated. The number of the BMC obtained from intact old animals increased faster in primary culture, than from young animals. The Cu induced fibrosis had different influence on the rate of BMC culture growth of old and young animals. The adding of 4 mM and 8 mM CuSO4x5H2O in the BMC culture of young and old animals resulted in a dose-dependent inhibition of growth rate of young animal cells. If copper ions were added into the culture of BMC of old animals, the decreased of the BMC number was described less than for cells of young animals. The adding of 8 mM CuSO4x5H2O inhibited proliferation less, than the adding of 4 mM CuSO4x5H2O. The Cu-induced liver fibrosis had accelerated the BMC rate death of both old and young animals. However, this effect was more pronounced in young animals. It is suggested, that during the ontogenesis the BMC undergo such epigenetic changes, which change functional properties.

Bone cells in cultures on nanocarbon-based materials for potential bone tissue engineering: A review

Czech Academy of Sciences Publication Activity Database

Bačáková, Lucie; Kopová, Ivana; Staňková, Ľubica; Lišková, Jana; Vacík, Jiří; Lavrentiev, Vasyl; Kromka, Alexander; Potocký, Štěpán; Stránská, D.

2014-01-01

Roč. 211, č. 12 (2014), s. 2688-2702 ISSN 1862-6300 R&D Projects: GA ČR(CZ) GAP108/12/1168; GA ČR(CZ) GA14-04790S; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 ; RVO:68378271 ; RVO:61389005 Keywords : biocompatibility * bone implants * carbon * nanoparticles Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.616, year: 2014

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

Directory of Open Access Journals (Sweden)

Jennifer Gordon

Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

Bis-enoxacin Inhibits Bone Resorption and Orthodontic Tooth Movement

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Toro, E.J.; Zuo, J.; Guiterrez, A.; La Rosa, R.L.; Gawron, A.J.; Bradaschia-Correa, V.; Arana-Chavez, V.; Dolce, C.; Rivera, M.F.; Kesavalu, L.; Bhattacharyya, I.; Neubert, J.K.; Holliday, L.S.

2013-01-01

Enoxacin inhibits binding between the B-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments, and also between osteoclast formation and bone resorption in vitro. We hypothesized that a bisphosphonate derivative of enoxacin, bis-enoxacin (BE), which was previously studied as a bone-directed antibiotic, might have similar activities. BE shared a number of characteristics with enoxacin: It blocked binding between the recombinant B-subunit and microfilaments and inhibited osteoclastogenesis in cell culture with IC50s of about 10 µM in each case. BE did not alter the relative expression levels of various osteoclast-specific proteins. Even though tartrate-resistant acid phosphatase 5b was expressed, proteolytic activation of the latent pro-enzyme was inhibited. However, unlike enoxacin, BE stimulated caspase-3 activity. BE bound to bone slices and inhibited bone resorption by osteoclasts on BE-coated bone slices in cell culture. BE reduced the amount of orthodontic tooth movement achieved in rats after 28 days. Analysis of these data suggests that BE is a novel anti-resorptive molecule that is active both in vitro and in vivo and may have clinical uses. Abbreviations: BE, bis-enoxacin; V-ATPase, vacuolar H+-ATPase; TRAP, tartrate-resistant acid phosphatase; αMEM D10, minimal essential media, alpha modification with 10% fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RANKL, receptor activator of nuclear factor kappa B-ligand; NFATc1, nuclear factor of activated T-cells; ADAM, a disintegrin and metalloprotease domain; OTM, orthodontic tooth movement. PMID:23958763

Diet and chronology of the neolithic cultures in the low povolzhye

DEFF Research Database (Denmark)

Vybornov, A. A.; Kulkova, Marianna A.; Kosintsev, Pavel

. The identification of animal bones allowed the detailed reconstruction of the meat component of the diet. Fish bones indicate the consumption of freshwater. Onager, saiga antelope, red deer, aurochs, wild-boar, corsac fox, wolf, birds and fish were found in Kairshak type sites of the Seroglazovskaya culture (Baybek......-boar were identified. Sheep and goats were determined as domestic animals. This is the first evidence for producing economy in Povolzhye. There are ten radiocarbon dates for this cultural type from 5500 to 4800 calBC. The AMS dates on the bones and organics from pottery have a good correlation...... these context have older ages. The results of lipid analysis of charred food crusts on the pottery from several sites showed that the meat and plant food prevailed in the diet of people in this region. These results allow for the first palaeodietary reconstructions of people of different cultures in the Low...

Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche.

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Templeton, Zach S; Lie, Wen-Rong; Wang, Weiqi; Rosenberg-Hasson, Yael; Alluri, Rajiv V; Tamaresis, John S; Bachmann, Michael H; Lee, Kitty; Maloney, William J; Contag, Christopher H; King, Bonnie L

2015-12-01

Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche. Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry. Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1β (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment. Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1β, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor.

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Ilchmann, Anne; Krause, Maren; Heilmann, Monika; Burgdorf, Sven; Vieths, Stefan; Toda, Masako

2012-05-01

The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove's modified Dulbecco's medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR) 2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly, BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4(+) T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture. Copyright © 2012 Elsevier Ltd. All rights reserved.

L-Carnitine halts apoptosis and myelosuppression induced by carboplatin in rat bone marrow cell cultures (BMC).

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Abd-Allah, Adel R A; Al-Majed, Abdulhakeem A; Al-Yahya, Abdulaziz A; Fouda, Soliman I; Al-Shabana, Othman A

2005-07-01

Carboplatin (CP), a second generation platinum compound, is effective against various types of cancers, producing less nephrotoxicity and ototoxicity but more myelotoxicity than cisplatinum. CP-myelosuppression is the rate-limiting step of its clinical use. Prevention of CP-myelosuppression is a major target in the field of chemotherapy. Therefore, the present study investigates the use of L-carnitine (LCR)-an antioxidant, cardioprotective, neuroprotective, and immunostimulant nontoxic natural compound-to protect against CP-induced myelosuppression. The viability of BMC was studied using a trypan blue exclusion technique following incubation with CP and/or LCR as a function of time and concentration. Apoptosis was tested for by detecting the amount of DNA fragmentation and the visualization of DNA ladders upon gel electrophoresis. Bone marrow progenitor cell function was examined by colony forming unit assay. Cellular contents of glutathione (GSH) and malondialdehyde (MDA) were also estimated. Results revealed that LC50 of CP is 4.7 mM and the highest safe concentration of LCR is 5 mM. Co-exposure of LCR+CP rescued BMC viability by 37% compared to the CP-treated cultures. The LCR halts CP-induced apoptosis and it significantly improves the function of the bone marrow progenitors by increasing the number of colony-forming units as a response to granulocyte/macrophage colony stimulating factors. Finally, LCR restores CP-induced GSH depletion and prevents MDA elevation in BMC. In summary, the results suggest that LCR is able to protect against CP-induced myelosuppression, which suggests its use as an adjuvant therapy. This finding merits further investigation into the mechanism(s) of such protection as well as its interaction with CP antitumor activity.

Bone marrow infection with mycobacterium fortuitum in a diabetic patient

International Nuclear Information System (INIS)

Satti, L.; Abbasi, S.; Sattar, A.; Ikram, A.; Manzar, M.A.; Khalid, M.M.

2011-01-01

Incidence and prevalence of Mycobacterium fortuitum infection vary greatly by location and death is very rare except in disseminated disease in immunocompromised individuals. We present what we believe is the first case of bone marrow infection with Mycobacterium fortuitum in an HIV negative patient. Bone marrow examination revealed presence of numerous acid fast bacilli which were confirmed as Mycobacterium fortuitum on culture and by molecular analysis. Patient was managed successfully with amikacin and ciprofloxacin. (author)

In vitro model of vascularized bone: synergizing vascular development and osteogenesis.

Directory of Open Access Journals (Sweden)

Cristina Correia

Full Text Available Tissue engineering provides unique opportunities for regenerating diseased or damaged tissues using cells obtained from tissue biopsies. Tissue engineered grafts can also be used as high fidelity models to probe cellular and molecular interactions underlying developmental processes. In this study, we co-cultured human umbilical vein endothelial cells (HUVECs and human mesenchymal stem cells (MSCs under various environmental conditions to elicit synergistic interactions leading to the colocalized development of capillary-like and bone-like tissues. Cells were encapsulated at the 1:1 ratio in fibrin gel to screen compositions of endothelial growth medium (EGM and osteogenic medium (OM. It was determined that, to form both tissues, co-cultures should first be supplied with EGM followed by a 1:1 cocktail of the two media types containing bone morphogenetic protein-2. Subsequent studies of HUVECs and MSCs cultured in decellularized, trabecular bone scaffolds for 6 weeks assessed the effects on tissue construct of both temporal variations in growth-factor availability and addition of fresh cells. The resulting grafts were implanted subcutaneously into nude mice to determine the phenotype stability and functionality of engineered vessels. Two important findings resulted from these studies: (i vascular development needs to be induced prior to osteogenesis, and (ii the addition of additional hMSCs at the osteogenic induction stage improves both tissue outcomes, as shown by increased bone volume fraction, osteoid deposition, close proximity of bone proteins to vascular networks, and anastomosis of vascular networks with the host vasculature. Interestingly, these observations compare well with what has been described for native development. We propose that our cultivation system can mimic various aspects of endothelial cell-osteogenic precursor interactions in vivo, and could find utility as a model for studies of heterotypic cellular interactions that

Tissue-engineered bone formation using human bone marrow stromal cells and novel β-tricalcium phosphate

International Nuclear Information System (INIS)

Liu Guangpeng; Zhao Li; Cui Lei; Liu Wei; Cao Yilin

2007-01-01

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel β-tricalcium phosphate (β-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous β-TCP scaffolds with pores of 300-500 μm in size were prepared by the polymeric sponge method. β-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous β-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In β-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in β-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous β-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering

Tissue-engineered bone formation using human bone marrow stromal cells and novel {beta}-tricalcium phosphate

Energy Technology Data Exchange (ETDEWEB)

Liu Guangpeng [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Zhao Li [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cui Lei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Liu Wei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cao Yilin [National Tissue Engineering Research and Development Center, Shanghai 200235 (China)

2007-06-01

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel {beta}-tricalcium phosphate ({beta}-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous {beta}-TCP scaffolds with pores of 300-500 {mu}m in size were prepared by the polymeric sponge method. {beta}-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous {beta}-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In {beta}-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in {beta}-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous {beta}-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

A feasibility study for in vitro evaluation of fixation between prosthesis and bone with bone marrow-derived mesenchymal stem cells.

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Morita, Yusuke; Yamasaki, Kenichi; Hattori, Koji

2010-10-01

It is difficult to quantitatively evaluate adhesive strength between an implant and the neighboring bone using animal experiments, because the degree of fixation of an implant depends on differences between individuals and the clearance between the material and the bone resulting from surgical technique. A system was designed in which rat bone marrow cells were used to quantitatively evaluate the adhesion between titanium alloy plates and bone plates in vitro. Three kinds of surface treatment were used: a sand-blasted surface, a titanium-sprayed surface and a titanium-sprayed surface coated with hydroxyapatite. Bone marrow cells obtained from rat femora were seeded on the titanium alloy plates, and the cells were cultured between the titanium alloy plates and the bone plates sliced from porcine ilium for 2 weeks. After cultivation, adhesive strength was measured using a tensile test, after which DNA amount and Alkaline phosphatase activity were measured. The seeded cells accelerated adhesion of the titanium alloy plate to the bone plate. Adhesive strength of the titanium-sprayed surface was lower than that of the sand-blasted surface because of lower initial contact area, although there was no difference in Alkaline phosphatase activity between two surface treatments. A hydroxyapatite coating enhanced adhesive strength between the titanium alloy palate and the bone plate, as well as enhancing osteogenic differentiation of bone marrow cells. It is believed that this novel experimental method can be used to simultaneously evaluate the osteogenic differentiation and the adhesive strength of an implant during in vitro cultivation. 2010 Elsevier Ltd. All rights reserved.

Use of NASA Bioreactor in Engineering Tissue for Bone Repair

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Duke, Pauline

1998-01-01

This study was proposed in search for a new alternative for bone replacement or repair. Because the systems commonly used in repair of bony defects form bone by going through a cartilaginous phase, implantation of a piece of cartilage could enhance the healing process by having a more advanced starting point. However, cartilage has seldom been used to replace bone due, in part, to the limitations in conventional culture systems that did not allow production of enough tissue for implants. The NASA-developed bioreactors known as STLV (Slow Turning Lateral Vessel) provide homogeneous distribution of cells, nutrients, and waste products, with less damaging turbulence and shear forces than conventional systems. Cultures under these conditions have higher growth rates, viability, and longevity, allowing larger "tissue-like" aggregates to form, thus opening the possibilities of producing enough tissue for implantation, along with the inherent advantages of in vitro manipulations. To assure large numbers of cells and to eliminate the use of timed embryos, we proposed to use an immortalized mouse limb bud cell line as the source of cells.