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1

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

PubMed

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

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

2013-02-01

2

BONE FORMATION INDUCED IN MOUSE THIGH BY CULTURED HUMAN CELLS  

PubMed Central

Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. 35S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible 35S utilization and secretion. FL cells, labeled in vitro with thymidine-3H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts.

Anderson, H. Clarke; Coulter, P. R.

1967-01-01

3

Microcarriers designed for cell culture and tissue engineering of bone.  

PubMed

Microspherical particulates have been an attractive form of biomaterials that find usefulness in cell delivery and tissue engineering. A variety of compositions, including bioactive ceramics, degradable polymers, and their composites, have been developed into a microsphere form and have demonstrated the potential to fill defective bone and to populate tissue cells on curved matrices. To enhance the capacity of cell delivery, the conventional solid form of spheres is engineered to have either a porous structure to hold cells or a thin shell to in-situ encapsulate cells within the structure. Microcarriers can also be a potential reservoir system of bioactive molecules that have therapeutic effects in regulating cell behaviors. Due to their specific form, advanced technologies to culture cell-loaded microcarriers are required, such as simple agitation or shaking, spinner flask, and rotating chamber system. Here, we review systematically, from material design to culture technology, the microspherical carriers used for the delivery of cells and tissue engineering, particularly of bone. PMID:23126371

Park, Jeong-Hui; Pérez, Román A; Jin, Guang-Zhen; Choi, Seung-Jun; Kim, Hae-Won; Wall, Ivan B

2013-04-01

4

Bioactive glass-ceramic containing crystalline apatite and wollastonite initiates biomineralization in bone cell cultures  

Microsoft Academic Search

Rat bone cells were cultured in the presence of bioactive glass-ceramic containing crystalline apatite and wollaston te. Scanning electron microscopy observations of the surface of the seeded ceramic disks revealed that cells attached, spread, and proliferated on the material surface. Soaking in cell-free culture medium showed that no change occurred in the surface structure. However, when cultured with bone cells

J. M. Sautier; T. Kokubo; T. Ohtsuki; J. R. Nefussi; H. Boulekbache; M. Oboeuf; S. Loty; C. Loty; N. Forest

1994-01-01

5

Spaceflight effects on cultured embryonic chick bone cells  

NASA Technical Reports Server (NTRS)

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

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

2000-01-01

6

Culture and characterization of human bone marrow mesenchymal stem cells.  

PubMed

Bone marrow (BM) mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of generating colonies of plastic-adherent marrow mesenchymal cells, each derived from a single cell termed a colony-forming unit fibroblasts (CFU-Fs). In addition to their role in establishing the marrow microenvironment, these cells have been shown to differentiate into several types of mesenchymal and non-mesenchymal lineages. Because of their multipotency, MSCs represent an attractive cellular source in the promising field of cellular therapy. In this chapter, we will focus on culture conditions for human BM MSC expansion and CFU-F assays. We also describe the methodologies to analyze the primary cultures obtained, both at the phenotypic and at the functional levels. Phenotypically, MSCs can be defined with a minimal set of markers as CD31-, CD34-, and CD45-negative cells and CD13-, CD29-, CD73-, CD90-, CD105-, and CD166-positive cells. Functionally, we describe the culture conditions (specific media and cellular preparations) for in vitro differentiation of MSCs into the adipogenic, osteogenic, and chondrogenic lineages. The corresponding colorimetric assays (oil red O, Von Kossa and alizarin red S, and safranin O and alcian blue stains, respectively) are also described. PMID:18085203

Delorme, Bruno; Charbord, Pierre

2007-01-01

7

Bone cell transfection in tissue culture using hydroxyapatite microparticles.  

PubMed

Coprecipitates of calcium phosphate and DNA have been used in vitro for several decades for cell transfection. We evaluated the efficiency of calcium phosphate ceramics associated to plasmid DNA in the transfection of bone cells in vitro when they are grown in tissue culture. Newborn rat calvariae and tibia epiphyses were grown on an agar surface for a period of 48 h to 30 days. The hydroxyapatite (HA)-particles were loaded with a plasmid bearing a galactosidase reporter gene by incubation of the plasmid solution in PBS with the particles. One milligram of HA-particles was then placed in contact with the bone explants for 8 and 30 days. Histological sections were then performed and the galactosidase activity was revealed using an X-gal solution. At eight days, very few cells expressing the galactosidase activity were detected. By 30 days, however, the explants appeared uniformly stained blue. The staining of sections showed that the osteoblasts, chondroblasts, perichondroblasts, and perisoteal cells all expressed the lacZ gene while the number of cells stained in the control was negligible. The time dependence of the transfection suggests that transfection using ceramics is linked to the degradation of the ceramic by the cells. Furthermore, the cells are stained remote from the particles suggesting that the particles induce a coprecipitate of DNA in the explant. PMID:16752398

Frayssinet, Patrick; Rouquet, Nicole; Mathon, Didier

2006-11-01

8

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

SciTech Connect

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

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

1991-08-01

9

[Modified method for whole bone marrow adherent culture of human bone marrow mesenchymal stem cells].  

PubMed

This study was aimed to investigate a more convenient and efficient method to cultivate the human bone marrow mesenchymal stem cells by means of natural erythrocyte sedimentation principle, based on the whole bone marrow adherent method. The bone marrow was cultured with a six-well plate instead of the flasks.Firsly, the bone marrow specimen was cultivated with the MSC complete medium for 48 h, then the upper RBC-free supernatant layer was drawn and placed into the new wells to isolate MSC. Inverted microscope was used to observe the cell morphology and to record the adherent time of first cell passage, first passaging time. The traditional whole bone marrow adherent method was used as the control. The cell cycle and cell surface markers were detected by flow cytometry,and the differentiative capacity of MSC into osteocyte and adipocyte was identified by alkaline phosphatase kit and oil red O, respectively. Besides, the proliferative curve of P1,P3,P5 of BMSC was depicted by counting method. The results showed that MSC cultured by the modified method highly expressed CD90, CD105, CD13, CD44 and lowly expressed CD14, CD45, CD34. Concerning the cell cycle feature, it was found that most of the cells were in G0/G1 phase (88.76%) , followed by G2/M phase (3.04%) and S phase (8.2%), which was in accordance with stem cell cycle characteristics. The proliferative curve showed a typical "S" type, and both the oil red O and alkaline phosphatase staining of MSC were positive. Compared with the traditional method, the modified method had the advantage of high adherence rate (P = 0.0001) and shorter passaging time for the first passage (P = 0.001), with the statistically significant difference. It is concluded that there is a large number of adherent, active and suspended MSC in the RBC-free supernatant layer after the culture of bone marrow for 48 h. Isolating MSC by the modified method is more convenient and efficient than the traditional whole bone marrow adherent method. PMID:24763030

Wang, Xiao-Qing; Zhong, Zhao-Dong; Chen, Zhi-Chao; Zou, Ping

2014-04-01

10

[A new method for isolating and culturing mouse bone marrow mesenchymal stem cells].  

PubMed

This study was purposed to establish a convenient and efficient method for isolating and culturing mouse bone marrow mesenchymal stem cells (MSC). The femurs and tibias of mouse were taken under sterile condition. MSC were isolated and cultured with flushing- out bone marrow or collagenase-digested bone fragment or bone marrow plus bone fragment. MSC colony number and size were compared. Immunophenotype and differentiation ability were tested to identify MSC. The results showed that colonies from bone marrow plus bone fragment group came out earliest and the colony number was 20 ± 4 at day 4; there were 11.5 ± 2.5 colonies in collagenase-digested bone fragment group and 9.5 ± 1.5 in flushing- out bone marrow group. The total cell yields of MSC after passaging showed best in bone marrow plus bone fragment group. Flow cytometry data showed the cultured cells expressed Sca-1, CD44 and CD29, not expressed pan-leukocyte surface marker CD45 and endothelial cell marker CD31. The isolated and cultured MSC could differentiate into osteoblast at the osteogenic differentiation condition, or adipocyte at adipogenic differentiation condition. It is concluded that the method of bone marrow plus bone fragment is convenient and efficient for isolating and culturing MSC. PMID:24370049

Yang, Yan-Mei; Li, Hong; Zhang, Lei; Dang, Rui-Jie; Li, Ping; Wang, Xiao-Yan; Zhu, Heng; Guo, Xi-Min; Zhang, Yi; Liu, Yuan-Lin; Mao, Ning; Jiang, Xiao-Xia; Wen, Ning

2013-12-01

11

Benefits of hypoxic culture on bone marrow multipotent stromal cells  

PubMed Central

Cultivation of cells is usually performed under atmospheric oxygen tension; however, such a condition does not replicate the hypoxic conditions of normal physiological or pathological status in the body. Recently, the effects of hypoxia on bone marrow multipotent stromal cells (MSCs) have been investigated. In a long-term culture, hypoxia can inhibit senescence, increase the proliferation rate and enhance differentiation potential along the different mesenchymal lineages. Hypoxia also modulates the paracrine effects of MSCs, causing upregulation of various secretable factors, including the vascular endothelial growth factor and interleukin-6, and thereby promoting wound healing and diabetic fracture healing. Finally, hypoxia plays an important role in mobilization and homing of MSCs, primarily by its ability to induce stromal cell-derived factor-1 expression along with its receptor, CXCR4. After transplantation, an ischemic environment, that is the combination of hypoxia and lack of nutrition, can lead to apoptosis or cell death, which can be overcome by the hypoxic preconditioning of MSCs and overexpression of prosurvival genes like Akt, HO-1 and Hsp70. This review emphasizes that hypoxia is an important factor in all major aspects of stem cell biology, and the mechanism involved in the hypoxic inducible factor-1signaling pathway behind these responses is also discussed.

Tsai, Chih-Chien; Yew, Tu-Lai; Yang, Der-Chi; Huang, Wei-Hua; Hung, Shih-Chieh

2012-01-01

12

Passage of bone-marrow-derived liver stem cells in a proliferating culture system  

Microsoft Academic Search

AIM: To explore the feasibility of passage of bone- marrow-derived liver stem cells (BDLSCs) in culture systems that contain cholestatic serum. METHODS: Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL\\/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors

Yun-Feng Cai; Ji-Sheng Chen; Shu-Ying Su; Zuo-Jun Zhen; Huan-Wei Chen

2009-01-01

13

Fatty acid oxidation in bone tissue and bone cells in culture. Characterization and hormonal influences.  

PubMed Central

Fatty acid oxidation and its hormonal modulation were investigated in cultured rat calvaria and in cultivated cell populations. The latter were obtained from calvaria of newborn rats by sequential time-dependent digestion with collagenase, yielding eight cell populations: the early ones containing mainly fibroblasts, the middle ones being osteoblast-like, and late ones osteoblast-osteocyte-like. In calvaria, fatty acid oxidation was increased by adding 0.1 mM- and 1.0 mM-palmitate to the medium, containing 10% (v/v) fetal-calf serum. No effect was found after parathyrin addition in vitro or when injected in vivo. All cell populations obtained by sequential digestion were found to oxidize palmitate, whereby the osteoblast-like cells showed a lower oxidation rate than the other populations. Both parathyrin and calcitonin had no effect on fatty acid oxidation. 1,25-Dihydroxycholecalciferol at 1-100 nM and 24,25-dihydroxycholecalciferol at 100 nM increased oxidation primarily in the population enriched with osteoblast-like cells. Insulin at 1.6 microM diminished it in the cell populations enriched with osteoblast-like cells and in the late bone-cell fraction. However, glucagon had no effect. The energy provided by fatty acid oxidation in this system is approx. 40-80% of glucose metabolism, suggesting that this event may be of importance in the energy metabolism of bone.

Adamek, G; Felix, R; Guenther, H L; Fleisch, H

1987-01-01

14

[A pilot study on the culture and differentiation of bone marrow stromal cells from SD rats].  

PubMed

In order to observe the growth, expansion and differentiation of the cultured bone marrow stromal cells (BMSC), we isolated the BMSC from adult SD rats and cultivated them with LIF and bFGF. Then, we cultured and induced the stem cells by using retinoic acid and the culture medium confected in our lab by ourselves. We found that the BMSC could expand and generate clones when they were cultured in vitro. These cells subcultured grew rapidly and differentiated into neuron-like cells and astrocyte-like cells. The results showed that BMSC have the abilities to self renew and differentiate, thus demonstrating the culture method we used is suitable for the culture of BMSC in vitro. The bone marrow stromal cell is not difficult to obtain; it is capable of expanding and differentiating in culture. If the culture condition is appropriate, it can differentiate into neuron and astrocyte. So, it is a kind of perfect seed cells. PMID:15022454

Li, Gang; Ke, Yiquan; Jiang, Xiaodan; Xu, Ruxiang; Zhou, Yuxi; Wang, Wei; Cheng, Wenping; Liao, Keli

2004-02-01

15

Characterization of conditioned medium of cultured bone marrow stromal cells  

Microsoft Academic Search

It has been recognized that bone marrow stromal cell (BMSC) transplantation has beneficial effects on spinal cord injury in animal models and therapeutic trials. It is hypothesized that BMSCs provide microenvironments suitable for axonal regeneration and secrete some trophic factors to rescue affected cells from degeneration. However, the molecular and cellular mechanisms of the trophic factors involved remain unclear. In

Norihiko Nakano; Yoshiyasu Nakai; Tae-Boem Seo; Yoshihiro Yamada; Takayuki Ohno; Atsuo Yamanaka; Yoji Nagai; Masanori Fukushima; Yoshiyuki Suzuki; Toshio Nakatani; Chizuka Ide

2010-01-01

16

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

PubMed Central

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

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

2010-01-01

17

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

Microsoft Academic Search

Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP\\/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP

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

2005-01-01

18

Isolation and culture of bone-forming cells (osteoblasts) from human bone.  

PubMed

The most conspicuous function of the osteoblast is the formation of bone. During phases of active bone formation, osteoblasts synthesize bone matrix and prime it for subsequent mineralization. Active osteoblasts are plump, cuboidal cells rich in organelles involved in the synthesis and secretion of matrix proteins. Unlike fibroblasts, they are obviously polarized, secreting matrix onto the underlying bony substratum which consequently grows by apposition. Some osteoblasts are engulfed in matrix during bone formation and are entombed in lacunae. These cells are described as osteocytes and remain in the bone matrix in a state of low metabolic activity. At the completion of a phase of bone formation, those osteoblasts which avoided entombment in lacunae lose their prominent synthetic function and become inactive osteoblasts, otherwise known as bone-lining cells. In mature bone, lining cells cover most of the bone surfaces. Osteocytes and bone-lining cells should not be considered as inactive cells since they play a major role in the regulation of bone modeling and remodeling and in calcium homeostasis (1). PMID:21359747

Gallagher, J A; Gundle, R; Beresford, J N

1996-01-01

19

Cultured human bone marrow-derived mast cells, their similarities to cultured murine E-mast cells.  

PubMed Central

Homogeneous populations of human mast cells were differentiated and grown by culturing bone marrow cells in the presence of conditioned medium derived from lectin-stimulated human peripheral blood mononuclear cells. The cells obtained were similar in ultrastructure, proteoglycan type and lipid products generated upon calcium ionophore A23187, and immunological activation to the murine E-mast cells (E-MC) differentiated in culture containing IL-3. Fluorescence analysis revealed that the human E-MC expressed IgE-Fc receptors which retained bound IgE through several washes. These cells did not express cell-surface lymphoid determinants (T11, T4, T8 and B4) and myeloid determinants 'My'. However, 40% of these cells expressed monocytic surface determinants, such as M-1. The amount of histamine that was found per 10(6) cells was 525 +/- 106 ng (mean +/- SE, n = 4). These cultured mast cells possessed granular chondroitin sulphate E proteoglycan of about 180,000 MW. Following activation with either calcium ionophore A23187 or anti-hIgE challenge, these mast cells released their preformed mediators and generated mainly leukotriene C4 leukotriene B4, and platelet-activating factor. In conclusion, according to all of these criteria, these human cultured mast cells show many similarities to the murine cultured E-mast cells, and therefore could be considered as the culture analogue of the human intestinal E-mast cells identified recently. Images Figure 1

Gilead, L; Rahamim, E; Ziv, I; Or, R; Razin, E

1988-01-01

20

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

PubMed

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

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

2013-12-01

21

Human bone tissue formation in diffusion chamber culture in vivo by bone-derived cells and marrow stromal fibroblastic cells.  

PubMed

Direct grafts of human cells into immunocompromised or cortisone-treated animals, either alone or within carrier materials, have been used with some success to assess the developmental capability of the grafted cells. However, identification of the donor or host origins of the generated tissue in such direct grafts is essential. In an alternative and extensively used experimental system, cells are cultured within the isolated environments of diffusion chambers, which are surgically implanted in appropriate hosts. This system allows the direct study of the cellular potentials for differentiation as host tissues are excluded. In the present study, human osteoprogenitor cell populations derived from trabecular bone explants or marrow suspensions of 3 patients (2 females aged 14 years and 1 male aged 27 years) were cultured in the absence or the continuous presence of dexamethasone (10 nmol/L). Cells were impregnated into porous hydroxyapatite ceramics before subcutaneous implantation, or placed within diffusion chambers before intraperitoneal implantation, in athymic mice. All subcutaneous implants of cells in ceramic showed morphological evidence for the formation of bone tissue. In the diffusion chambers it was found that both marrow- and bone-derived fibroblastic cells cultured in the absence of dexamethasone generally produced fibrous tissue only. When cultured in the continuous presence of dexamethasone (10 nmol/L), these cell populations produced similar osteogenic tissues with active osteoblasts, wide osteoid seams, and mineralized tissue, with cartilage toward the interior of the chamber. These results validate the diffusion chamber as an experimental system to study human osteogenesis using appropriately primed cell populations. PMID:7669435

Gundle, R; Joyner, C J; Triffitt, J T

1995-06-01

22

Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells.  

PubMed

Cells showing osteoclastic characteristics have not been identified outside bone. Because osteoclasts originate from an extraosseous source, this suggests that identifiable osteoclastic features do not develop until the precursors enter bone, where the local microenvironment may signal osteoclastic differentiation or maturation. We assessed the influence of bone matrix on osteoclastic differentiation by incubating bone marrow cells, after removal of pre-existing osteoclasts, on plastic coverslips or slices of devitalized cortical bone. We found that there was a threefold increase in the number of osteoclast-specific MAb-positive cells on the bone matrix compared with plastic coverslips. The number of MAb-positive cells correlated with the extent of excavation of the surface of the bone slices. Multinuclearity correlated with MAb-positive cell density, and for any given density the proportion of MAb-positive cells that were multinucleate was similar on plastic and bone. We conclude that, in the presence of 1,25-(OH)2 vitamin D3, bone matrix stimulates the generation of osteoclasts but has no demonstrable influence on the fusion of mononuclear osteoclastic precursors. PMID:2728923

Fuller, K; Chambers, T J

1989-04-01

23

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

Microsoft Academic Search

Summary  By culturing bone marrow mesenchymal stem cells of rabbits with fibrin gluein vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue\\u000a engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4–6 ml of bone marrow were aspirated from\\u000a rabbit femoral trochanter. The monocytes suspension was aspirated

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

2004-01-01

24

Generation and characterization of bone marrow-derived cultured canine mast cells  

Microsoft Academic Search

Disorders of mast cells, particularly mast cell tumors (MCTs), are common in dogs. There now is evidence that many of these disorders exhibit breed predilections, suggesting an underlying heritable component. In comparison to humans and mice, little is known regarding the biology of canine mast cells. To facilitate the study of mast cell biology in other species, bone marrow-derived cultured

Tzu-yin Lin; Laura J. Rush; Cheryl A. London

2006-01-01

25

A functional comparison of canine and murine bone marrow derived cultured mast cells  

Microsoft Academic Search

Disorders involving mast cells are extremely common in dogs, ranging from allergic diseases to neoplastic transformation resulting in malignant mast cell tumors. Relatively little is known regarding the basic biologic properties of normal canine mast cells, largely due to the difficulty in reliably purifying large numbers from canine skin. In vitro generated bone marrow derived cultured mast cells (BMCMCs) are

Tzu-Yin Lin; Cheryl A. London

2006-01-01

26

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

PubMed Central

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

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

2014-01-01

27

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

PubMed Central

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse.

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

2011-01-01

28

Effects of Laddec reg; on the formation of calcified bone matrix in rat calvariae cells culture  

Microsoft Academic Search

Osteoblasts from 21 day-old fetal rats calvaria were isolated using a collagenase digestion procedure. Cells were cultured in the presence of Laddec® (a highly purified bovine xenograft) and Bio-Oss® (natural bone mineral). Optical microscopic observations showed that osteoblasts attached on the plastic culture dishes and formed close contact with biomaterial particles. By day 5, the osteoblasts formed a confluent monolayer.

S. Hofman; M. Sidqui; D. Abensur; P. Valentini; P. Missika

1999-01-01

29

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

PubMed Central

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

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

2013-01-01

30

Perfusion Enhances Functions of Bone Marrow Stromal Cells in Three-Dimensional Culture  

Microsoft Academic Search

Perfusion of medium through three-dimensional (3D) collagen sponges enhanced viability and function of cocultivated marrow stromal and hematopoietic cell lines. Cells of the murine bone marrow stromal cell line GPIa were cultured in novel 3D collagen sponges, made from pepsin-digested bovine skin. Static cultures of sponges were maintained in dishes with media changes every other day. Perfused sponges were contained

Julie Glowacki; Shuichi Mizuno; Joel S Greenberger

1998-01-01

31

Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture  

Microsoft Academic Search

Basic fibroblast growth factor (bFGF) was shown to enhance rat stromal bone marrow cells in culture to produce mineralized bone-like tissue in response to dexamethasone (Dex) treatment (Pitaru et al., J Bone Miner Res 8:919; 1993). The purpose of this study was to explore the effect of bFGF on Dex-treated human stromal bone marrow cells (hSBMC) in culture. Human SBMC

S Pri-Chen; S Pitaru; F Lokiec; N Savion

1998-01-01

32

Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells.  

PubMed

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody-positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2 vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2 vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody-positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues. PMID:3308907

Fuller, K; Chambers, T J

1987-09-01

33

Bone culture research  

NASA Technical Reports Server (NTRS)

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.

Partridge, Nicola C.

1993-01-01

34

Scleractinium Coral Aquaculture Skeleton: a Possible 3D Scaffold for Cell Cultures and Bone Tissue Engineering.  

PubMed

Cytocompatibility of 5 coral aquaculture skeleton species derived from two families (Acroporidae and Pocilloporidae) was studied over the course of in vitro culturing in continuous human fibroblast culture by the MMT test. Biocompatibility and capacity of scaffold to "transfer" cell cultures (specifically, multipotent mesenchymal stromal cells) to sites of implantation were studied in vivo by subcutaneous implantation of skeletal fragments to rats. All coral skeleton aquaculture specimens were cytocompatible (nontoxic and with surface matrix characteristics satisfactory for cells), biocompatible, and could be tried as 3D matrices for bone tissue engineering. PMID:24771438

Sergeeva, N S; Britaev, T A; Sviridova, I K; Akhmedova, S A; Kirsanova, V A; Popov, A A; Antokhin, A I; Frank, G A; Kaprin, A D

2014-02-01

35

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

SciTech Connect

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

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

1985-05-01

36

Regulation of BMP-Induced Transcription in Cultured Human Bone Marrow Stromal Cells  

PubMed Central

Background Adherent bone marrow stromal cells are inducible osteoprogenitors, giving rise to cells expressing osteoblast markers including alkaline phosphatase, osteopontin, osteocalcin, and bone sialoprotein. However, the potency of inducers varies in a species-specific manner. Glucocorticoids such as dexamethasone induce alkaline phosphatase activity in both human and rat mesenchymal stem cells, while mouse bone marrow stromal cells are refractory to dexamethasone-induced alkaline phosphatase activity. In contrast, BMP induces alkaline phosphatase activity in both mouse and rat bone marrow stromal cells, while BMP effects on human bone marrow stromal cells are poorly characterized. Methods Bone marrow samples were isolated from patients undergoing hip replacement. Mononuclear marrow cells were cultured and grown to confluence without or with 10?7M dexamethasone. Cells from each isolate were passaged into medium containing 100 ?g/mL ascorbate phosphate and treated with dexamethasone, 100 ng/mL BMP, or no inducer. At day 6, alkaline phosphatase activity was assayed, and RNA was prepared for mRNA analyses by real-time polymerase chain reaction. Results Bone marrow stromal cells from twenty-four of twenty-six patients showed no significant osteogenic response to BMP-2, 4, or 7 as determined by alkaline phosphatase induction. However, BMPs induced elevated levels of other genes associated with osteogenesis such as bone sialoprotein and osteopontin as well as BMP-2 and noggin. If primary cultures of human bone marrow stromal cells were pretreated with dexamethasone, BMP-2 treatment of first-passage cells induced alkaline phosphatase in approximately half of the isolates, and significantly greater induction was seen in cells from males. Dexamethasone treatment, like BMP treatment, also increased expression of the BMP-binding protein noggin. Conclusions Most human femur bone marrow stromal cell samples appear incapable of expressing elevated alkaline phosphatase levels in response to BMPs. Since BMP treatment induced expression of several other BMP-regulated genes, the defect in alkaline phosphatase induction is presumably not due to impaired BMP signaling. We hypothesize that the mechanism by which BMPs modulate alkaline phosphatase expression is indirect, involving a BMP-regulated transcription factor for alkaline phosphatase expression that is controlled differently in humans and rodents. Clinical Relevance We suggest that the relative insensitivity of alkaline phosphatase to BMP induction in human bone marrow stromal cells may contribute to the variation in efficacy reported with BMP in clinical settings.

Diefenderfer, David L.; Osyczka, Anna M.; Garino, Jonathan P.; Leboy, Phoebe S.

2005-01-01

37

Comparisons of Rabbit Bone Marrow Mesenchymal Stem Cell Isolation and Culture Methods In Vitro  

PubMed Central

Bone marrow mesenchymal stem cells (BMSCs) have great potential in tissue engineering and clinical therapy, and various methods for isolation and cultivation of BMSCs have been reported. However, the best techniques are still uncertain. Therefore, we sought the most suitable among the four most common methods for BMSC separation from rabbits. BMSCs were obtained from untreated whole bone marrow (BM) adherent cultures, 3 volumes of red blood cells (RBC) lysed with ammonium chloride, 6 volumes of RBC lysed with ammonium chloride, and Ficoll density gradient centrifugation. Then, isolated BMSCs were evaluated with respect to primary cell yield, number of CFU-F colonies, proliferative capacity, cell phenotype, and chondrogenic differentiation potential. Our data show that BMSCs were successfully isolated by all four methods, and each method was similar with regard to cell morphology, phenotype, and differentiation potential. However, BMSCs from untreated whole BM adherent cultures had greater primary cell yields, larger colonies, and the shortest primary culture time (P<0.05). Moreover, the 4th generation of cultured cells had the strongest proliferative activity, the fastest growth rate and the most numerous cells compared with other cell passage generations (P<0.05). In conclusion, untreated whole BM adherent cultures are best for rabbit BMSC isolation and the 4th generation of cells has the strongest proliferation capacity.

Zhang, Weidong; Zhang, Fangbiao; Shi, Hongcan; Tan, Rongbang; Han, Shi; Ye, Gang; Pan, Shu; Sun, Fei; Liu, Xingchen

2014-01-01

38

Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro.  

PubMed

Bone marrow mesenchymal stem cells (BMSCs) have great potential in tissue engineering and clinical therapy, and various methods for isolation and cultivation of BMSCs have been reported. However, the best techniques are still uncertain. Therefore, we sought the most suitable among the four most common methods for BMSC separation from rabbits. BMSCs were obtained from untreated whole bone marrow (BM) adherent cultures, 3 volumes of red blood cells (RBC) lysed with ammonium chloride, 6 volumes of RBC lysed with ammonium chloride, and Ficoll density gradient centrifugation. Then, isolated BMSCs were evaluated with respect to primary cell yield, number of CFU-F colonies, proliferative capacity, cell phenotype, and chondrogenic differentiation potential. Our data show that BMSCs were successfully isolated by all four methods, and each method was similar with regard to cell morphology, phenotype, and differentiation potential. However, BMSCs from untreated whole BM adherent cultures had greater primary cell yields, larger colonies, and the shortest primary culture time (P<0.05). Moreover, the 4(th) generation of cultured cells had the strongest proliferative activity, the fastest growth rate and the most numerous cells compared with other cell passage generations (P<0.05). In conclusion, untreated whole BM adherent cultures are best for rabbit BMSC isolation and the 4(th) generation of cells has the strongest proliferation capacity. PMID:24558428

Zhang, Weidong; Zhang, Fangbiao; Shi, Hongcan; Tan, Rongbang; Han, Shi; Ye, Gang; Pan, Shu; Sun, Fei; Liu, Xingchen

2014-01-01

39

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

SciTech Connect

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.

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

1990-08-01

40

Attachment of human bone cells to tissue culture polystyrene and to unmodified polystyrene: the effect of surface chemistry upon initial cell attachment  

Microsoft Academic Search

Cell culture studies have often been used in the determination of the suitability of biomaterials as surfaces for the attachment and growth of cells. For such studies of surfaces for potential use in bone implants, cells derived from bone may be maintained in culture on tissue culture polystyrene (TCPS). We have determined the contribution that serum fibronectin (FN) or vitronectin

John G. Steele; Clive McFarland; B. Ann Dalton; Graham Johnson; Margaret D. M. Evans; C. Rolfe Howlett; P. Anne Underwood

1994-01-01

41

Histological and cytological technique for the quantitation of cultured human bone-marrow cells: Formation of aggregates  

Microsoft Academic Search

Summary  A bone-marrow culture system is described that provides a simple, quantitative and rapid assessment of marrow bone cells in\\u000a vitro. Aggregation of bone-marrow cells, an in vitro phenomenon, occurs within 24 hr of culture and is observed utilizing\\u000a Millipore filters. Daily quantitation shows both an increase in the number and a change in the morphology of these aggregates.\\u000a The maximum

Vincent F. Garry; Mark Nesbitt; James White; Gerald Vosika

1978-01-01

42

[Isolation of mesenchymal stem cells from bone marrow filters by primary explant culture].  

PubMed

This study was aimed to investigate whether mesenchymal stem cells (MSC) can be isolated from bone marrow filters which have always been discarded. The bone marrow (BM) particles from BM filters of 2 healthy donors were cultivated by primary explant culture. After expansion, the number of MSC was counted and their immunophenotype and differentiation potential were detected. The results indicated that many MSC were found in bone marrow particles from filters, and nearly 10(7) MSC were obtained at 3 passages of expansion. They not only possessed the characteristics of morphology and immunophenotype of MSC, but also could differentiate into osteoblasts, chondrocytes and adipocytes. In conclusion, a large amount of MSC can be obtained from BM filters if the BM particles were cultivated by primary explant culture. PMID:21518508

Xing, Wen; Yang, Shao-Guang; Liu, Meng; Lu, Shi-Hong; Zhao, Qin-Jun; Pang, Ai-Ming; Yao, Jian-Feng; Li, Jian-Ping; Ren, Qian; Han, Zhong-Chao

2011-04-01

43

A practical guide to culturing mouse and human bone marrow stromal cells.  

PubMed

Bone marrow stromal cells (BMSCs, frequently also called MSCs) represent a cell population within the bone marrow, a subset of which contains multipotent stem cells. Their primary role is to produce and maintain both bone tissue and bone marrow microenvironment necessary for hematopoiesis. The latter is achieved by secreting a wide variety of different cytokines and growth factors, many of which also have a regulatory role in immune processes. BMSCs have recently been introduced into the field of immunobiology after their successful clinical use in GVHD was reported in 2004. Since then, numerous studies confirmed and expanded the knowledge on the immunosuppressive potential of BMSCs in various in vitro and in vivo models. Although the immunomodulatory capacity of BMSCs is well established, there are still many unanswered questions regarding the cytokines, chemokines, receptors, and molecular pathways that play a role in this effect. To study these cells and answer many of the questions, researchers must be able to reliably and reproducibly isolate, culture, and use these cells. Below a practical guide on how to culture and characterize mouse and human BMSCs, which can then be applied in various in vitro and in vivo assays, is provided. PMID:24510517

Nemeth, K; Mayer, B; Sworder, B J; Kuznetsov, S A; Mezey, E

2013-01-01

44

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

SciTech Connect

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

Fletcher, K.

1981-01-01

45

Cytocidal infection of hog cholera virus in porcine bone marrow stroma cell cultures.  

PubMed

Porcine bone marrow stroma cell (BMSC) cultures producing cells of granulocyte-lineage were established. Hog cholera (HC) virus ALD and Alfort strains replicated in the porcine BMSC cultures showing distinct cytopathic effect (CPE). The differentiation of granulocyte-lineage cells in the cultures ceased after infection with HC virus. Polyclonal antibody against the ALD strain inhibited completely the development of CPE of the both ALD and Alfort strains. Monoclonal antibodies (mAbs) specific to the ALD strain inhibited CPE of the ALD strain, while CPE of the Alfort strain was not affected by those mAbs, suggesting that CPE induced in the BMSC cultures is due to HC virus. PMID:8748554

Shimizu, M; Yamada, S; Nishimori, T

1995-12-01

46

Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study.  

PubMed

Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering. PMID:24652549

Jin, Guang-Zhen; Park, Jeong-Hui; Seo, Seog-Jin; Kim, Hae-Won

2014-07-01

47

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

PubMed

In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds (Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy. PMID:20703817

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

2011-11-01

48

Histological and cytological technique for the quantitation of cultured human bone-marrow cells:formation of aggregates.  

PubMed

A bone-marrow culture system is described that provides a simple, quantitative and rapid assessment of marrow bone cells in vitro. Aggregation of bone-marrow cells, an in vitro phenomenon, occurs within 24 hr of culture and is observed utilizing Millipore filters. Daily quantitation shows both an increase in the number and a change in the morphology of these aggregates. The maximum number of aggregates is achieved on the 2nd or 3rd day of incubation. Histologically, aggregates are composed of myeloid, mononuclear and mesenchymal fibroblastic cells. Mesenchymal cells form a matrix for apposed mononuclear and myeloid cells. Scanning electron micrographs show intimate cell contact and spreading by the marrow cells. Fluctuation of the absolute numbers of various cell types are observed. The system can be utilized for long-term culture of bone marrow. PMID:680770

Garry, V F; Nesbitt, M; White, J; Vosika, G

1978-06-01

49

Response of osteoblast-like cells cultured on zirconia to bone morphogenetic protein-2  

PubMed Central

Purpose The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2. Methods MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days. Results At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7. Conclusions The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.

Han, Seung-Hee; Kim, Kyoung-Hwa; Han, Jung-Seok; Koo, Ki-Tae; Kim, Tae-Il; Seol, Yang-Jo; Lee, Yong-Moo; Ku, Young

2011-01-01

50

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

SciTech Connect

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

Rentala, Satyanarayana [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067 (India); Sagar Balla, Murali Mohan [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067 (India); Khurana, Satish [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067 (India); Mukhopadhyay, Asok [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067 (India)]. E-mail: asok@nii.res.in

2005-09-30

51

Bone tissue engineering of induced pluripotent stem cells cultured with macrochanneled polymer scaffold.  

PubMed

A reliable source of osteogenic cells is an essential factor for bone tissue engineering. In this study, human-induced pluripotent stem cells (hiPSCs) without an embryoid body step were cultured in macrochanneled poly(caprolactone) (PCL) scaffolds prepared using a robotic dispensing technique, after which osteogenesis was promoted by the addition of exogenous osteogenic factors. The osteogenesis of the hiPSCs was demonstrated based on the detection of osteogenic molecules, such as osteopontin, using flow cytometry analysis, quantitative polymerase chain reaction and western blotting. Thereafter, the cell-scaffold constructs were transplanted into the subcutaneous site of male athymic mice. At 4 weeks after implantation, histological assays (hematoxylin & eosin staining, Alizarin red staining, and osteocalcin immunostaining) were conducted to determine the bone induction of hiPSCs. The results indicated a production of pronounced levels of extracellular matrices and their mineral deposition within the cell-scaffold implant, suggesting possible in vivo bone induction by the hiPSCs-based tissue engineering approach. The results presented here provide useful information regarding the tissue engineering of bone utilizing hiPSCs in conjunction with cell-supporting scaffolds. PMID:23065721

Jin, Guang-Zhen; Kim, Tae-Hyun; Kim, Joong-Hyun; Won, Jong-Eun; Yoo, So-Young; Choi, Seong-Jun; Hyun, Jung Keun; Kim, Hae-Won

2013-05-01

52

Three-Dimensional Cancer-Bone Metastasis Model Using Ex-Vivo Co-Cultures of Live Calvarial Bones and Cancer Cells  

PubMed Central

One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free floating live mouse calvarial bone organs in the presence of cancer cells in a roller-tube system. In such co-cultures under hypoxia and a specifically defined bone remodeling stage, viz., resorption system, cancer cells showed a remarkable affinity and specificity for the “endosteal side” of the bone where they colonize and proliferate. This was concurrent with differentiation of resident stem/progenitor cells to osteoclasts and bone resorption. In contrast, under bone formation conditions this model revealed different pathophysiology where the breast cancer cells continued to induce osteoclastic bone resorption whereas prostate cancer cells led to osteoblastic bone formation. The current 3D model was used to demonstrate its application to studies involving chemical and biochemical perturbations in the absence and presence of cancer cells and cellular responses. We describe proof-of-principle with examples of the broad versatility and multi-faceted application of this model that adds another dimension to the ongoing studies in the cancer-bone metastasis arena.

Curtin, Paul; Youm, Helen; Salih, Erdjan

2011-01-01

53

Enhanced differentiation of adult bone marrow-derived stem cells to liver lineage in aggregate culture.  

PubMed

Hepatocyte-like cells derived from stem cells hold great potential for clinical and pharmaceutical applications, including high-throughput drug toxicity screening. We report a three-dimensional aggregate culture system for the directed differentiation of adult rat bone marrow-derived stem cells, rat multipotent adult progenitor cells, to hepatocyte-like cells. Compared to adherent monolayer cultures, differentiation in the aggregate culture system resulted in significantly higher expression level of liver-specific transcripts, including an increased albumin mRNA level, and higher levels of albumin and urea secretion. This coincides with the presence of significantly more cells that express intracellular albumin at levels found in primary hepatocytes. The differentiated cell aggregates exhibited cytochrome P450-mediated ethoxyresorufin-O-dealkylation and pentoxyresorufin-O-dealkylation activity. Consistent with these increased mature functions, cells within the aggregates were shown to have many ultrastructural features of mature hepatocytes by transmission electron microscopy. With the scalability of the aggregate culture system and the enhanced differentiation capability, this system may facilitate translation of generating hepatocytes from stem cells to technology. PMID:21548835

Subramanian, Kartik; Owens, Derek Jason; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

2011-09-01

54

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

PubMed

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

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

2014-09-01

55

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

SciTech Connect

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.

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

1986-07-15

56

[Efficient isolation of mesenchymal stem cells from human bone marrow by direct plating method combined with modified primary explant culture].  

PubMed

Human bone marrow is the major source of mesenchymal stem cells (MSC). It was reported that the standard density gradient centrifugation method was not efficient in isolating MSC and it may be caused by the existing of bone marrow particles. In previous study, a lot of MSC were obtained by culturing bone marrow particles alone combined with standard method. However, it is time- and labor-consuming to obtain bone marrow particles by filtering and to isolate MNC by density gradient centrifugation. This study was purposed to explore the more simple and efficient method to isolate MSC from bone marrow. Seven normal bone marrow aspirates were collected and centrifugated. The bone marrow particles floated on surface layers were cultured by modified primary explant culture, whereas the bone marrow aspirates deposited were cultured by direct plating method, then the immun phenotype and differentiation capability of isolated cells were analyzed. The results showed that in 3 of 7 aspirates, bone marrow particles were floated on surface layers, whereas the other bone marrow cells and some particles were deposited after centrifugation. The MSC were reliably isolated from the floating layers or deposited aspirates by modified primary explant culture and direct plating method separately. After 3 passages the isolated MSC did not express CD45 and CD34, but expressed CD105, CD73, CD44, CD90, CD49e and they could differentiate into chondrocytes and adipocytes. It is concluded that normal human bone marrow MSC can be isolated simply and efficiently by direct plating method in combination with modified primary explant culture. PMID:23628052

Xing, Wen; Pang, Ai-Ming; Yao, Jian-Feng; Li, Yuan; Shi, Hui; Sheng, Meng-Yao; Zhou, Yuan; Zhao, Ying-Xu; Xu, Ming-Jiang; Yang, Feng-Chun

2013-04-01

57

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures.  

PubMed

Thyroid hormones (T3,T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links. T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality. PMID:20707983

Varga, F; Rumpler, M; Zoehrer, R; Turecek, C; Spitzer, S; Thaler, R; Paschalis, E P; Klaushofer, K

2010-11-12

58

Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells.  

PubMed

Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation. PMID:23011741

Li, Xueyuan; Zhang, Yang; Qi, Guoxian

2013-05-01

59

Strain-specific variations in the development of dendritic cells in murine bone-marrow cultures.  

PubMed

The dendritic cell (DC) is a professional antigen-presenting cell of central importance in immunity. In this paper, we examined DCs generated by 11-day culture of bone-marrow cells from the four mouse strains C57BL/6J, BALB/cA, C3H/HeN and B10.PL-H2u (73NS)/Sn with respect to cell yield as well as surface-marker phenotype and morphology. We also investigated the phenotypic changes and the T-cell stimulatory activity of the DCs induced by bacterial lipopolysaccharide (LPS). Morphologically, we observed low levels (5-10%) of granulocyte contamination of the cultures after a culture period of 11 days. Considerable strain-specific differences were found in the expression levels of the surface markers in addition to the differences in the ratio of the immature to mature DCs in the cultures that were not stimulated with LPS. Furthermore, we found that LPS strongly induces maturation of DCs in all strains investigated with the exception of the B10.PL strain. PMID:10849369

Petersen, M S; Toldbod, H E; Holtz, S; Hokland, M; Bolund, L; Agger, R

2000-06-01

60

Generation and characterization of bone marrow-derived cultured canine mast cells.  

PubMed

Disorders of mast cells, particularly mast cell tumors (MCTs), are common in dogs. There now is evidence that many of these disorders exhibit breed predilections, suggesting an underlying heritable component. In comparison to humans and mice, little is known regarding the biology of canine mast cells. To facilitate the study of mast cell biology in other species, bone marrow-derived cultured mast cells (BMCMCs) often are used because these represent a ready source of large numbers of cells. We have developed a protocol to successfully generate canine BMCMCs from purified CD34(+) cells. After 5-7 weeks of culture with recombinant canine stem cell factor (rcSCF), greater than 90% of the cell population consisted of mast cells as evidenced by staining with Wright's-Giemsa, as well as production of chymase, tryptase, IL-8 and MCP-1. These cells expressed cell surface markers typical of mast cells including Kit, Fc epsilonRI, CD44, CD45 and CD18/CD11b. The canine BMCMCs were dependent on rcSCF for survival and proliferation, and migrated in response to rcSCF gradients. Cross-linking of cell surface-bound IgE induced the release of histamine and TNFalpha. Histamine release could also be stimulated by ConA, compound 48/80, and calcium ionophore. In summary, canine BMCMCs possess phenotypic and functional properties similar to mast cells found in vivo. These cells represent a novel, valuable resource for investigating normal canine mast cell biology as well as for identifying factors that lead to mast cell dysregulation in the dog. PMID:16780961

Lin, Tzu-yin; Rush, Laura J; London, Cheryl A

2006-09-15

61

Matrix formation is enhanced in co-cultures of human meniscus cells with bone marrow stromal cells.  

PubMed

The ultimate aim of this study was to assess the feasibility of using human bone marrow stromal cells (BMSCs) to supplement meniscus cells for meniscus tissue engineering and regeneration. Human menisci were harvested from three patients undergoing total knee replacements. Meniscus cells were released from the menisci after collagenase treatment. BMSCs were harvested from the iliac crest of three patients and were expanded in culture until passage 2. Primary meniscus cells and BMSCs were co-cultured in vitro in three-dimensional (3D) pellet culture at three different cell-cell ratios for 3 weeks under normal (21% O2 ) or low (3% O2 ) oxygen tension in the presence of serum-free chondrogenic medium. Pure BMSCs and pure meniscus cell pellets served as control groups. The tissue generated was assessed biochemically, histochemically and by quantitative RT-PCR. Co-cultures of primary meniscus cells and BMSCs resulted in tissue with increased (1.3-1.7-fold) deposition of proteoglycan (GAG) extracellular matrix (ECM) relative to tissues derived from BMSCs or meniscus cells alone under 21% O2 . GAG matrix formation was also enhanced (1.3-1.6-fold) under 3% O2 culture conditions. Alcian blue staining of generated tissue confirmed increased deposition of GAG-rich matrix. mRNA expression of type I collagen (COL1A2), type II collagen (COL2A1) and aggrecan were upregulated in co-cultured pellets. However, SOX9 and HIF-1? mRNA expression were not significantly modulated by co-culture. Co-culture of primary meniscus cells with BMSCs resulted in increased ECM formation. Co-delivery of meniscus cells and BMSCs can, in principle, be used in tissue engineering and regenerative medicine strategies to repair meniscus defects. PMID:22473741

Matthies, Norah-Faye; Mulet-Sierra, Aillette; Jomha, Nadr M; Adesida, Adetola B

2013-12-01

62

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.  

PubMed

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. PMID:16798036

Simão, Ana Maria S; Beloti, Márcio M; Cezarino, Rodrigo M; Rosa, Adalberto Luiz; Pizauro, João M; Ciancaglini, Pietro

2007-04-01

63

Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture.  

PubMed

Basic fibroblast growth factor (bFGF) was shown to enhance rat stromal bone marrow cells in culture to produce mineralized bone-like tissue in response to dexamethasone (Dex) treatment (Pitaru et al., J Bone Miner Res 8:919; 1993). The purpose of this study was to explore the effect of bFGF on Dex-treated human stromal bone marrow cells (hSBMC) in culture. Human SBMC from 6 patients were cultured for 14 days (P0) and then subcultured and grown for 28 days in the presence of Dex (10(-8) mol/L). The effect of bFGF on cell proliferation at P0 and protein content, DNA content, alkaline phosphatase activity (ALP), osteocalcin secretion, and formation of mineralized bone-like tissue (MBT) at P1 was analyzed. bFGF treatment resulted in a 2.4-fold increase in cell number at P0 and a concentration-dependent increase in [3H]-thymidine incorporation at P1, reaching a maximum increase of 3.7-fold at a concentration of 0.3 ng/mL. Furthermore, bFGF significantly increased both DNA content (two- to threefold), protein content (five- to sixfold), and the amount of MBT (up to 20-fold) at P1 cultures. Morphological evaluation of the MBT at the electron microscope level revealed a mineralization process along collagen fibrils similar to the natural process. The osteogenic nature of the bFGF-treated cultures was further shown by their ALP activity, as well as osteocalcin secretion in response to 1,25-dihydroxyvitamin D3. In conclusion, bFGF demonstrated a stimulatory effect on the proliferation of Dex-treated hSBMC-derived osteoprogenitors while maintaining their capacity to fully differentiate and form bone-like tissue in culture. PMID:9701469

Pri-Chen, S; Pitaru, S; Lokiec, F; Savion, N

1998-08-01

64

Osteoblast differentiation of bone marrow stromal cells cultured on silica gel and sol–gel-derived titania  

Microsoft Academic Search

Primary cultures of osteogenic precursor cells derived from rat bone marrow stroma were performed on commercially available pure titanium discs (Ti c.p.) and surface modified Ti c.p.using a sol–gel technique (Ti sol). In separate repeated experimental runs, cell behavior and in vitro mineralization were compared with cultures on silica gel bioactive glass discs (S53P4). All substrates were incubated in simulated

S. C Dieudonné; J van den Dolder; J. E de Ruijter; H Paldan; T Peltola; M. A van ’t Hof; R. P Happonen; J. A Jansen

2002-01-01

65

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

PubMed Central

The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.

2010-01-01

66

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

Microsoft Academic Search

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

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

1979-01-01

67

The Impact of Cell Source, Culture Methodology, Culture Location, and Individual Donors on Gene Expression Profiles of Bone Marrow-Derived and Adipose-Derived Stromal Cells  

PubMed Central

Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

Prins, Henk-Jan; Schrama, Ellen; Verwiel, Eugene T.P.; Martens, Anton C.M.; Roelofs, Helene; Jansen, Bastiaan J.H.

2013-01-01

68

Oncornavirus-Like Particles from Cultured Bone Marrow Cells Preceding Leukemia and Malignant Histiocytosis  

Microsoft Academic Search

Particles with the density and enzymatic activity characteristic of known oncornavirus have been previously described in bone marrow cells from patients with leukemia in relapse and in remission. We have confirmed these findings and studied two patients in whom preleukemia was among the diagnostic considerations. Following cultivation of bone marrow from these patients for 1 week in conditioned media with

Gerald J. Vosika; William Krivit; Jon M. Gerrard; Peter F. Coccia; Mark E. Nesbit; Jacqueline J. Coalson; B. J. Kennedy

1975-01-01

69

Effect of Ex Vivo Culture of CD34+ Bone Marrow Cells on Immune Reconstitution of XSCID Dogs Following Allogeneic Bone Marrow Transplantation  

PubMed Central

Successful genetic treatment of most primary immunodeficiencies or hematological disorders will require the transduction of pluripotent, self-renewing hematopoietic stem cells (HSC) rather than their progeny in order to achieve enduring production of genetically corrected cells and durable immune reconstitution. Current ex vivo transduction protocols require manipulation of HSC by culture in cytokines for various lengths of time depending upon the retroviral vector that may force HSC to enter pathways of proliferation, and possibly differentiation, that could limit their engraftment potential, pluripotentiality and long-term repopulating capacity. We have compared the ability of normal CD34+ cells cultured in a standard cytokine cocktail for 18 hours or 4.5 days to reconstitute XSCID dogs following bone marrow transplantation in the absence of any pre-transplant conditioning with that of freshly isolated CD34+ cells. CD34+ cells cultured under standard ?-retroviral transduction conditions (4.5 days) showed decreased engraftment potential and ability to sustain long-term thymopoiesis. In contrast, XSCID dogs transplanted with CD34+ cells cultured for 18 hours showed a robust T cell immune reconstitution similar to dogs transplanted with freshly isolated CD34+ cells, however, the ability to sustain long-term thymopoiesis was impaired. These results emphasize the need to determine ex vivo culture conditions that maintain both the engraftment potential and “stem cell” potential of the cultured cells.

Kennedy, Douglas R.; McLellan, Kyle; Moore, Peter F.; Henthorn, Paula S.; Felsburg, Peter J.

2009-01-01

70

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

PubMed

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 ?1 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 further our understanding of the PCa-bone crosstalk. PMID:24530694

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

2014-06-01

71

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow  

Microsoft Academic Search

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5×106 cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e.,

Manfred B Lutz; Nicole Kukutsch; Alexandra L. J Ogilvie; Susanne Rößner; Franz Koch; Nikolaus Romani; Gerold Schuler

1999-01-01

72

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

Microsoft Academic Search

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

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

2010-01-01

73

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells.  

PubMed

One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion techniques were compared: (1) CD133+ selection followed by ex vivo liquid culture and (2) co-culture of unmanipulated CB with bone-marrow-derived mesenchymal stem cells (MSCs). Ex vivo culture was performed in medium supplemented with granulocyte colony-stimulating factor, stem cell factor and either thrombopoietin or megakaryocyte growth and differentiation factor. Expansion was followed by measuring total nucleated cell (TNC), CD133+ and CD34+ cell, colony-forming unit and cobblestone area-forming cell output. When compared to liquid culture, CB-MSC co-culture (i) required less cell manipulation resulting in less initial HPC loss and (ii) markedly improved TNC and HPC output. CB-MSC co-culture therefore holds promise for improving engraftment kinetics in CB transplant recipients. PMID:16400333

Robinson, S N; Ng, J; Niu, T; Yang, H; McMannis, J D; Karandish, S; Kaur, I; Fu, P; Del Angel, M; Messinger, R; Flagge, F; de Lima, M; Decker, W; Xing, D; Champlin, R; Shpall, E J

2006-02-01

74

Long-Term Erythropoiesis from Constant Numbers of CD34 + Cells in Serum-free Cultures Initiated with Highly Purified Progenitor Cells from Human Bone Marrow  

Microsoft Academic Search

Summary To directly study the biological properties of purified hematopoietic colony-forming cell precursors, cells with a CD34 + CD45RA l~ CD71 I~ phenotype were purified from human bone marrow using density separation and fluorescence-activated call sorting, and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3), Ib6, erythropoietin, and mast cell growth

Peter M. Lansdorp; Wieslawa Dragowska

75

Development of robotic dispensed bioactive scaffolds and human adipose-derived stem cell culturing for bone tissue engineering.  

PubMed

Bioactive and degradable scaffolds made from bioactive glass-polycaprolactone with a mineralized surface and a well-defined three-dimensional (3D) pore configuration were produced using a robotic dispensing technique. Human adipose-derived stem cells (hASCs) were cultured on the 3D scaffolds, and the osteogenic development of cells within the scaffolds was addressed under a dynamic flow perfusion system for bone tissue engineering. The bioactive glass component introduced within the composite assisted in the surface mineralization of the 3D scaffolds. The hASCs initially adhered well and grew actively over the mineralized surface, and migrated deep into the channels of the 3D scaffold. In particular, dynamic perfusion culturing helped the cells to proliferate better on the 3D structure compared to that under static culturing condition. After 4 weeks of culturing by dynamic perfusion, the cells not only covered the scaffold surface completely but also filled the pore channels bridging the stems. The osteogenic differentiation of the hASCs with the input of osteogenic factors was stimulated significantly by the dynamic perfusion flow, as determined by alkaline phosphate expression. Overall, the culturing of hASCs upon the currently developed 3D scaffold in conjunction with the dynamic perfusion method may be useful for tissue engineering of bone. PMID:19722827

Oh, Chung-Hun; Hong, Seok-Jung; Jeong, Ishik; Yu, Hye-Sun; Jegal, Seung-Hwan; Kim, Hae-Won

2010-08-01

76

Osteogenic differentiation of bone mesenchymal stem cells regulated by osteoblasts under EMF exposure in a co-culture system.  

PubMed

This study examined the osteogenic effect of electromagnetic fields (EMF) under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells (BMSCs) and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit (CCK-8). For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase (ALP) staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure (2 h/day) could obviously promote proliferation of BMSCs and osteoblasts, while long-term EMF (8 h/day) could promote osteogenic differentiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when cells were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differentiation of BMSCs. PMID:24710940

Yu, Ji-Zhe; Wu, Hua; Yang, Yong; Liu, Chao-Xu; Liu, Yang; Song, Ming-Yu

2014-04-01

77

Effect of high glucose on extensive culturing of mesenchymal stem cells derived from subcutaneous fat, omentum fat and bone marrow.  

PubMed

Frontline research progresses the applicability of bone marrow and adipose tissue in regenerative medicine, but fails to account for the functional improvement of the diseased. The justification for the failure in terms of stem cell survival, proliferation and regeneration is unclear. However, hyperglycemia rising during pathological conditions might be one such stumbling block. The prevailing literature accounts for both detrimental and beneficial effect of high glucose on mesenchymal stem cells (MSCs) leading to perplexity. Thus, this study focuses on the effect of high glucose on mesenchymal stem cells derived from subcutaneous fat, omentum fat and bone marrow in extensive cultures. We provide evidence for the retention of MSC characteristics of all sources with regards to surface marker profiling, proliferation, differentiation and karyotyping when cultured extensively under DMEM-HG containing glucose concentration of 25 mmol.l(-1) . Thus, it can be concluded that hyperglycemia in vivo (11 mmol.l(-1) ) might not be a barrier for the ineffective functional improvement of transplanted stem cells. Furthermore, we elucidated subcutaneous and omentum fat as better sources of MSCs when compared with bone marrow, thereby making these sources optimal for therapies during hyperglycemic conditions. However, further research is needed to clear the path for efficient stem cell transplantation. PMID:22729714

Dhanasekaran, M; Indumathi, S; Rajkumar, J S; Sudarsanam, D

2013-01-01

78

Stimulation of liver functions in hierarchical co-culture of bone marrow cells and hepatocytes  

Microsoft Academic Search

A hierarchial co-culture, in which rat hepatocytes and non-parenchymal liver cells (NPLCs) were separated by a collagen layer and which was designed to mimic the in vivo microenvironment, was carried out with the aim of developing a module for bio-artificial liver support. Compared with a monolayer co-culture and hepatocytes cultured alone in a monolayer, higher urea synthesis activity was maintained

Kiyohito Yagi; Nobuaki Sumiyoshi; Yumiko Nakashima; Nobuyasu Michibayashi; Masaya Kawase; Yoshiharu Miura; Tadashi Mizoguchi

1998-01-01

79

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

Microsoft Academic Search

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

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

80

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

PubMed Central

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

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

2010-01-01

81

Characterization of Human Bone Marrow Long-Term Cultures  

Microsoft Academic Search

In an attempt to more closely simulate the native hematopoietic environment, human bone marrow bony matrix was cultivated in long-term human bone marrow cultures. Sternal bone marrow curettings (i.e., ‘bony matrix’) were cultured with and without autologous bone marrow single cell suspensions. Fresh media were provided at weekly intervals and the ‘harvested’ cells were assayed for CFU-gm (i.e., the granulocyte-macrophage

Akira Horikoshi

1984-01-01

82

Cell therapy for bone repair.  

PubMed

When natural bone repair mechanisms fail, autologous bone grafting is the current standard of care. The osteogenic cells and bone matrix in the graft provide the osteo-inductive and osteo-conductive properties required for successful bone repair. Bone marrow (BM) mesenchymal stem cells (MSCs) can differentiate into osteogenic cells. MSC-based cell therapy holds promise for promoting bone repair. The amount of MSCs available from iliac-crest aspirates is too small to be clinically useful, and either concentration or culture must therefore be used to expand the MSC population. MSCs can be administered alone via percutaneous injection or implanted during open surgery with a biomaterial, usually biphasic hydroxyapatite/?-calcium-triphosphate granules. Encouraging preliminary results have been obtained in patients with delayed healing of long bone fractures or avascular necrosis of the femoral head. Bone tissue engineering involves in vitro MSC culturing on biomaterials to obtain colonisation of the biomaterial and differentiation of the cells. The biomaterial-cell construct is then implanted into the zone to be treated. Few published data are available on bone tissue engineering. Much work remains to be done before determining whether this method is suitable for the routine filling of bone tissue defects. Increasing cell survival and promoting implant vascularisation are major challenges. Improved expertise with culturing techniques, together with the incorporation of regulatory requirements, will open the way to high-quality clinical trials investigating the usefulness of cell therapy as a method for achieving bone repair. Cell therapy avoids the drawbacks of autologous bone grafting, preserving the bone stock and diminishing treatment invasiveness. PMID:24411717

Rosset, P; Deschaseaux, F; Layrolle, P

2014-02-01

83

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

PubMed

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

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

2014-01-01

84

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

PubMed Central

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

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

2014-01-01

85

Microgravity and bone cell mechanosensitivity  

NASA Astrophysics Data System (ADS)

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

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

2003-10-01

86

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

NASA Technical Reports Server (NTRS)

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

Lawless, Brother Desales

1990-01-01

87

Recombinant human bone morphogenetic protein-2 promotes osteogenesis within atelopeptide type I collagen solution by combination with rat cultured marrow cells.  

PubMed

We evaluated the combination effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and cultured rat bone marrow mesenchymal stem cells (MSCs) in atelopeptide type I collagen (AC) solution on osteogenesis in a diffusion chamber (DC) to develop a bone substitute having consistent osteogenic capability for clinical applications. The cultured MSCs were obtained by 10-day primary culture of fresh bone marrow cells of Fischer rats. We prepared three groups of DCs: AC solution with rhBMP-2, AC solution with cultured MSCs, and AC solution with rhBMP-2 and cultured MSCs. The prepared combined solutions were injected into DCs, which were subcutaneously implanted into the backs of syngeneic rats. DCs were harvested after 2, 4, or 8 weeks and analyzed for bone-forming capability by determining histological and osteoblastic biochemical markers. De novo bone formation was observed both inside and outside of the membrane filter of DCs in the group of AC solution with rhBMP-2 and cultured MSCs. The alkaline phosphatase activity and osteocalcin content in the group of AC solution with rhBMP-2 and cultured MSCs were significantly higher than those in the group of AC solution with cultured MSCs at any time. These findings indicate that AC aqueous solution is a useful material not only as a carrier of rhBMP-2 but also as a cell-anchorage for differentiation and proliferation of MSCs. Therefore, this study suggests that clinical repairs of bone defects are feasible using injectable AC solution with rhBMP-2 and cultured MSCs as a bone substitute. PMID:11835160

Ikeuchi, Masako; Dohi, Yoshiko; Horiuchi, Katsuhiro; Ohgushi, Hajime; Noshi, Toshiaki; Yoshikawa, Takafumi; Yamamoto, Kazuhiko; Sugimura, Masahito

2002-04-01

88

Microgravity and Bone Cell Mechanosensitivity  

NASA Astrophysics Data System (ADS)

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

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

89

Normal bone marrow adherent cell-conditioned medium corrects the impaired differentiation of cultured mononuclear phagocytes from vitamin D-deficient rats  

Microsoft Academic Search

Summary  There is increasing evidence that 1,25 dihydroxyvitamin D3 acts directly to differentiate and activate cells of mononuclear phagocyte lineage (MNP). However, it seems possible that\\u000a in bone marrow this hormone may modulate the differentiation of MNP via the factor elaborated by the stromal cells or the\\u000a macrophages. We tested this hypothesis using two separate culture systems of bone marrow cells

Takashi Nakamura; Kazuyuki Araki; Shigenobu Kanda; Kojiro Kurisu

1986-01-01

90

Influence of culture medium on smooth muscle cell differentiation from human bone marrow-derived mesenchymal stem cells.  

PubMed

Human bone marrow-derived mesenchymal stem cells (hMSCs) represent an appealing source of smooth muscle cells (SMCs) for engineering small-diameter vascular grafts due to the limited availability and replicative capacity of somatic SMCs. However, lack of standardization of hMSC culture conditions has limited some progress in hMSC research. Because, at the moment, a chemically defined, serum-free medium without growth factors is not capable of amplifying hMSCs in vitro, the usage of serum (either human serum or fetal bovine serum [FBS]) continues in hMSC research. The emergence of commercial hMSCs and hMSC media opened a series of questions regarding the compatibility of commercial and homemade hMSCs and hMSC media. In this study, two types of commonly used FBS-containing hMSC media-MSCGM (containing 10% FBS) and MesenPro (containing 2% FBS), along with our homemade medium (low-glucose Dulbecco's modified Eagle's medium plus 10% selected lot FBS)-were compared in their ability to support SMC differentiation from hMSCs. The effects of FBS level, medium supplements (ascorbic acid, copper, etc.), and growth factors (transforming growth factor beta1) were also examined for their impact on SMC differentiation. It was discovered that MesenPro and transforming growth factor beta1 are the strongest SMC inducers from hMSCs. In contrast, hMSCs grown in homemade (10% Dulbecco's modified Eagle's medium) and commercial MSCGM media remained undifferentiated. FBS concentration did not affect SMC differentiation when 10% FBS was compared with 2%. Finally, the mechanism underlying SMC differentiation from hMSCs grown in FBS-containing medium was explored by following the expression changes of serum response factor during the establishment of hMSC culture. PMID:19115826

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

2009-02-01

91

A Comparative Study on Morphochemical Properties and Osteogenic Cell Differentiation within Bone Graft and Coral Graft Culture Systems  

PubMed Central

The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system.

Puvaneswary, Subramaniam; Balaji Raghavendran, Hanumantha Rao; Ibrahim, Nurul Syuhada; Murali, Malliga Raman; Merican, Azhar Mahmood; Kamarul, T.

2013-01-01

92

Oncornavirus-like particles from cultured bone marrow cells preceding leukemia and malignant histiocytosis.  

PubMed

Particles with the density and enzymatic activity characteristic of known oncornavirus have been previously described in bone marrow cells from patients with leukemia in relapse and in remission. We have confirmed these findings and studied two patients in whom preleukemia was among the diagnostic considerations. Following cultivation of bone marrow from these patients for 1 week in conditioned media with dexamethasone, a high-speed pellet of the supernatant fluid and disrupted cells was prepared and analyzed on a sucrose gradient for enzymatic activity characteristic of RNA-directed DNA polymerase (reverse transcriptase). Peaks of endogenous DNA polymerase activity showing ribonuclease sensitivity and/or stimulation with the synthetic template poly(rC)-(dG)12-18 were demonstrated in both patients at densities of 1.15 to 1.19 and 1.21 to 1.24 g/ml. Subsequently, diagnosis 2 and 4 months after initial evaluation revealed acute myelogenous leukemia and malignant histiocytosis, respectively. Prior studies have suggested a possible etiological significance of such particles in human leukemia. The demonstration of similar particles preceding clinically overt disease in these patients supports this hypothesis and offers the possibility of early diagnosis and treatment. PMID:52158

Vosika, G J; Krivit, W; Gerrard, J M; Coccia, P F; Nesbit, M E; Coalson, J J; Kennedy, B J

1975-07-01

93

Oncornavirus-like particles from cultured bone marrow cells preceding leukemia and malignant histiocytosis.  

PubMed Central

Particles with the density and enzymatic activity characteristic of known oncornavirus have been previously described in bone marrow cells from patients with leukemia in relapse and in remission. We have confirmed these findings and studied two patients in whom preleukemia was among the diagnostic considerations. Following cultivation of bone marrow from these patients for 1 week in conditioned media with dexamethasone, a high-speed pellet of the supernatant fluid and disrupted cells was prepared and analyzed on a sucrose gradient for enzymatic activity characteristic of RNA-directed DNA polymerase (reverse transcriptase). Peaks of endogenous DNA polymerase activity showing ribonuclease sensitivity and/or stimulation with the synthetic template poly(rC)-(dG)12-18 were demonstrated in both patients at densities of 1.15 to 1.19 and 1.21 to 1.24 g/ml. Subsequently, diagnosis 2 and 4 months after initial evaluation revealed acute myelogenous leukemia and malignant histiocytosis, respectively. Prior studies have suggested a possible etiological significance of such particles in human leukemia. The demonstration of similar particles preceding clinically overt disease in these patients supports this hypothesis and offers the possibility of early diagnosis and treatment.

Vosika, G J; Krivit, W; Gerrard, J M; Coccia, P F; Nesbit, M E; Coalson, J J; Kennedy, B J

1975-01-01

94

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

PubMed

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

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

2014-01-01

95

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

PubMed Central

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

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

2014-01-01

96

Calcium-mediated enhancement of the cyclic AMP response in cultured bone cells.  

PubMed

We have examined the influence of extracellular Ca2+ on cyclic AMP metabolism in an osteoblast-enriched population of bone cells isolated from the calvaria of rat fetuses. The cyclic AMP response to stimulators of cyclic AMP formation (PTH and PGE2), but not basal cyclic AMP levels, increased progressively as the extracellular Ca2+ concentration was raised from 0.2 to 4.0 mM. The response to changes in extracellular Ca2+ were rapid (within 3.5 min), and the level of responsivity that characterized each Ca2+ concentration persisted for at least 6 h when the Ca2+ concentration was kept constant. The effect of Ca2+ spanned the entire time course of PTH action, was not accompanied by altered excretion of cyclic AMP from the cells, and was evident at low as well as at high hormone concentrations. Ca2+ augmented the action of PTH in the presence as well as in the absence of cyclic AMP phosphodiesterase inhibitors, and failed to decrease cyclic AMP phosphodiesterase activity in the short term. Mn2+ and, to a smaller degree, Ba2+ substituted for Ca2+ in promoting the cyclic AMP response to PTH. Verapamil, an inhibitor of Ca2+ penetration, blunted the Ca2+-mediated increments in the cyclic AMP response, and the divalent cation ionophore A23187 enhanced these increments. These results indicate that Ca2+ and other cations are positive effectors of the stimulated cyclic AMP response in isolated bone cells. Accumulation into an as yet unknown cellular compartment may be required for the cation effect. The data are most consistent with enhancement of adenylate cyclase reactivity as the mode of cation action. PMID:6271356

Peck, W A; Kohler, G; Barr, S

1981-01-01

97

Differentiation of human bone marrow-derived mesenchymal stem cells into neural-like cells by co-culture with retinal pigmented epithelial cells  

PubMed Central

AIM To detect the differentiation effects of retinal cells or extracts on bone marrow-derived mesenchymal stem cells (BMSC). METHODS Human fetal BMSC were previously labelled by carboxyfluorescein succinimidyl ester (CFSE), and co-cultured with retinal pigment epithelial (RPE) cells which were pre-treated with ultraviolet irradiation at a ratio of 1:1 to induce the differentiation of BMSC for up to 14 days. In some assays, a retinal extract of bovine retinal extract (BRE) was added to detect the potential effects of retinal component on the differentiation of BMSC. In addition, Neuron-specific enolase (NSE), Nestin and Glial fibrillary acidic protein (GFAP) immunostaining were performed to determine the characteristics of BMSC. RESULTS The results indicated that by co-cultured with RPE cells, fetal BMSC were differentiated into neural-like cells expressing special neuronal markers Nestin, GFAP and NSE. And the expression of these markers was obviously increased by BRE. CONCLUSION Retina derived cells and extracts can induce the differentiation of BMSC into neural-like cells.

Yang, Ling-Ling; Zhou, Qing-Jun; Wang, Yao; Wang, Yi-Qiang

2010-01-01

98

Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture.  

PubMed

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM-MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier-based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM-MSCs in stirred suspension bioreactors using CultiSpher-S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post-inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM-MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200-fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM-MSCs in culture, which in turn should facilitate the development of new stem cell-based therapies. PMID:22689330

Yuan, Yifan; Kallos, Michael S; Hunter, Christopher; Sen, Arindom

2014-03-01

99

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages  

PubMed Central

Background Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity. Design and Methods Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed. Results Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34+ cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34+ cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture. Conclusions Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.

Briquet, Alexandra; Dubois, Sophie; Bekaert, Sandrine; Dolhet, Marie; Beguin, Yves; Gothot, Andre

2010-01-01

100

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

SciTech Connect

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.

Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. (Osaka Univ. Medical School (Japan))

1991-03-01

101

Abnormal rearrangements of T-cell receptor genes occur in long-term cultured bone marrow cells of lpr/lpr mice.  

PubMed Central

To search the abnormality in prethymic T-cell precursors in lpr/lpr(lpr) mice, rearrangement and expression of T-cell receptor (TcR) genes were investigated in long-term cultured bone marrow (LTBM) cells of lpr mice, in which the developmental steps of T-cell precursors may be better synchronized than those in bone marrow (BM) cells. Neither rearrangment nor expression of TCR gamma and delta genes were detected in the LTBM cells from +/+ control mice, whereas some gamma gene rearrangements were detected in those derived from lpr mice, irrespective of the genetic background. When BM cells or LTBM cells from lpr mice were transplanted into supralethally irradiated +/+ mice the lpr-derived BM cells appeared earlier in the thymus of the recipient mice than +/+-derived BM cells and the recipients suffered from lethal wasting syndrome. In addition, the lpr-derived BM cells showed higher activity in colony-forming unit spleen (CFUs) than the +/+-derived BM cells. These results suggest that the T-cell progenitors in the BM of lpr mice may be different not only in quantity but also in quality from those of +/+ mice. Images Figure 1

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

1989-01-01

102

Adult Rat Bone Marrow-Derived Stem Cells Promote Late Fetal Type II Cell Differentiation in a Co-Culture Model  

PubMed Central

Bronchopulmonary dysplasia develops in preterm infants due to a combination of lung immaturity and lung injury. Cultured pluripotent bone marrow stem cells (BMSC) are known to reduce injury and induce repair in adult and in immature lungs, possibly through paracrine secretion of soluble factors. The paracrine relationship between BMSC and primary fetal lung epithelial type II cells is unknown. We determined the effects of BMSC on type II cell and fibroblast behavior using an in vitro co-culture model. Rat BMSC were isolated and co-cultured with primary fetal E21 rat type II cells or lung fibroblasts in a Transwell® system without direct cell contact. Effects of BMSC conditioned media (CM) on type II cell and fibroblast proliferation and on type II cell surfactant phospholipid (DSPC) synthesis and mRNA expression of surfactant proteins B and C (sftpb and sftpc) were studied. We also determined the effect of fibroblast and type II cell CM on BMSC proliferation and surface marker expression. Co-culture with BMSC significantly decreased type II cell and fibroblast proliferation to 72.5% and 83.7% of controls, respectively. Type II cell DSPC synthesis was significantly increased by 21% and sftpb and sftpc mRNA expressions were significantly induced (2.1 fold and 2.4 fold, respectively). BMSC proliferation was significantly reduced during the co-culture. Flow cytometry confirmed that BMSC retained the expression of undifferentiated stem cell markers despite their exposure to fetal lung cell CM. We conclude that BMSC induce fetal type II cell differentiation through paracrine release of soluble factors. These studies provide clues for how BMSC may act in promoting alveolar repair following injury.

Knoll, AB; Brockmeyer, T; Chevalier, R; Zscheppang, K; Nielsen, HC; Dammann, CE

2013-01-01

103

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

PubMed Central

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

2013-01-01

104

Enhanced cellular responses of human bone marrow stromal cells cultured on pretreated surface with allogenic platelet-rich plasma.  

PubMed

The principal objective of this study was to evaluate the effects of surface pretreatment with platelet-rich plasma (PRP) on the cellular functions of human bone marrow stromal cells (hBMSCs). The surfaces of tissue culture plates (TCPs) were pretreated by adding PRP followed by centrifugation to bring platelets closer to the surface, followed by incubation for 60 min at 37°C. Then, hBMSCs were seeded onto TCP and TCP pretreated with PRP (TCP-PRP), followed by culture in osteogenic medium. Cell attachment, proliferation, and osteogenic differentiation were evaluated. Field emission scanning electron microscope (FE-SEM; JSM-7401F, JEOL Ltd., Japan) observations were conducted. The attachment of hBMSCs was significantly lower on TCP-PRP than on TCP. However, when the cell numbers were normalized with those observed on day 1 of culture, cellular proliferation on 5 days was significantly higher on TCP-PRP. Alkaline phosphatase activity, an index of early phase of osteoblastic differentiation, was significantly higher on TCP-PRP on day 14. Calcium deposition amount, an index of terminal osteoblastic differentiation, was also significantly higher on TCP-PRP on days 14 and 21. The results of von Kossa staining confirmed that, on day 21, the area of mineralized nodules was significantly larger on TCP-PRP. FE-SEM observation demonstrated that activated platelets and fibrin network covered the surface after PRP treatment. An increase in the number of hBMSCs and their cellular products was evident on the FE-SEM observation, and the fibrin network remained on day 21. Our results demonstrate that a PRP-treated surface enhanced early proliferation and late osteogenic differentiation of hBMSCs. PMID:22329757

Shin, Seung Han; Yoo, Jeong Joon; Kim, Ha Na; Nam, Jinwoo; Kim, Hee Joong

2012-01-01

105

Bone tissue engineering with human stem cells  

Microsoft Academic Search

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

Darja Marolt; Miomir Knezevic; Gordana Vunjak Novakovic

2010-01-01

106

The Effect of Initial Cell Seeding Density on Early Osteogenic Signal Expression of Rat Bone Marrow Stromal Cells Cultured on Crosslinked Poly(propylene fumarate) Disks  

PubMed Central

The intercellular signaling mechanisms among a transplanted cell population are largely determined by the cell population itself as well as the surrounding environment. Changes in cell-to-cell paracrine signaling distance can be obtained by altering cell density, and signal expression of growth factors can be enhanced by auto/paracrine signal transduction. To examine these relationships, we investigated the effect of cell seeding density on viability, proliferation, differentiation, and the endogenous osteogenic signal expression among rat bone marrow stromal cells (BMSCs) cultured on a 2D disk. Rat BMSCs were isolated from rats and then cultured for 8 days on biodegradable poly(propylene fumarate) disks with three different seeding densities (0.06, 0.15, and 0.30 million cells/disk). At day 1, 4, and 8, viability by Live/Dead fluorescent staining, DNA amount, osteogenic differentiation by alkaline phosphatase and osteocalcin mRNA expression, calcium deposition, and osteogenic growth factor mRNA expression were assayed. Osteogenic signal expression was evaluated using quantitative reverse transcriptase-polymerase chain reaction, and signals of interest include bone morphogenetic protein-2, transforming growth factor-?1, fibroblast growth factor-2, and platelet-derived growth factor-A. The results from this study demonstrate that rat BMSCs were viable over 8 days without being affected by cell density as well as cell proliferation rate and early osteogenic differentiation were stimulated by lower cell seeding density. Most importantly, this study has demonstrated for the first time that the temporal gene expression profiles of endogenous growth factors can be controlled by altering the initial cell seeding density on poly(propylene fumarate) disks. Therefore, our result suggest that changes in the paracrine signal distance by altering cell seeding density may be a useful strategy to optimize the cell-biomaterial construct microenvironments to enhance the osteogenic signal expression.

Kim, Kyobum; Dean, David; Mikos, Antonios G.; Fisher, John P.

2013-01-01

107

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

Microsoft Academic Search

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

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

2007-01-01

108

New vitamin D less-calcemic analog affect human bone cell line and cultured vascular smooth muscle cells similar to other less-calcemic analogs.  

PubMed

Primary cultures of human bone and vascular cells respond to vitamin D treatment by modulation of cell proliferation measured by DNA synthesis (DNA) and energy metabolism measured by creatine kinase specific activity (CK) via binding to vitamin D receptors (VDR) which are expressed in these cells. Vitamin D compounds also modulate the response to estradiol-17? (E?) and the expression mRNAs of estrogen receptors (ER? and ER?), VDR, 25-hydroxy vitamin D? 1-? hydroxylase (1OHase) and lipoxygenases (12LO and 15LO). We now compared our newly synthesized analog: 1?,25-dihydroxy-9-methylene-19-norvitamin D? JK152 (JK), on bone and vascular cells compared to other analogs. Human bone cell line SaOS? respond to JK by increased DNA and stimulated CK dose-dependently, similar to the less-calcemic analogs CB 1093 (CB) and EB 1089 (EB). JK also up-regulated the response to E? in terms of DNA and CK. JK inhibited DNA synthesis and increased CK in primary human vascular smooth muscle cells (VSMC) dose-dependently similar to EB and CB. JK up regulated the response to E? in terms of CK with no effect on DNA. JK similar to CB and EB stimulated mRNA expression of VDR and ER?, 12LO and 15LO, with no effect on ER? and 1OHase mRNA expression in SaOS? measured by real time PCR. Similar treatments of VSMC with JK, CB and EB stimulated 12LO and 15LO, VDR and ER? mRNA expression with no effect on ER? and 1OHase mRNA expression. The results presented here demonstrate that the new vitamin D less-calcemic analog JK is similar to other analogs in its effects on human cultured cells and therefore may be used in combined hormone replacement treatment (HRT) both in vitro and in vivo. PMID:24269661

Somjen, D; Kulesza, U; Sharon, O; Knoll, E; Stern, N

2014-03-01

109

Enhanced osteogenesis of human alveolar bone-derived mesenchymal stem cells for tooth tissue engineering using fluid shear stress in a rocking culture method.  

PubMed

This study instituted a simple approach to stimulate alveolar bone regeneration for tooth tissue engineering by controlling effects of low fluid dynamic shear stress (LFDSS) on growth and differentiation in vitro. Human alveolar bone-derived mesenchymal stem cells (hABMSCs) harvested from human mandibular alveolar bone were cultured with LFDSS to generate cultures containing bone-like formations. To distinguish between osteodifferentiation and bone-like formation, cells were cultured either with or without fluid shear stress. The calcium content and alkaline phosphatase (ALP) activity of hABMSCs were used as indicators of osteogenesis. Cell viability and proliferation after stimulating with LFDSS for 10-60?min/day were higher than with longer stimulations. Mineralized nodules formed when osteoblasts were cultured with an induction medium, a marker of osteogenic differentiation. ALP activity tended to increase after 10 and 60?min/day of stimulation. In addition, LFDSS conditions also increased gene expression of IBSP, RUNX2, COL-I, ALP, OCN, and OPN, as shown by reverse transcriptase-polymerase chain reaction. From the results of a proteomics array, LFDSS groups were intensely expressed with several factors (EGF, HGF, IGF, TGF, and PDGF). Furthermore, CD146 and Stro-1 expression increased in cells treated with 30?min/day and decreased in cells treated with 120?min/day, as determined by cell surface antigen analysis by fluorescence-activated cell-sorting analysis. These results strongly showed that LFDSS at the proper intensity and time enhanced the differentiation and maturation of hABMSCs. In conclusion, an appropriate level of LFDSS can potently and positively modulate proliferation and differentiation in hABMSCs. PMID:23088630

Lim, Ki-Taek; Kim, Jangho; Seonwoo, Hoon; Chang, Jung Uk; Choi, Hwajung; Hexiu, Jin; Cho, Woo Jae; Choung, Pill-Hoon; Chung, Jong Hoon

2013-02-01

110

A novel ex vivo culture model for inflammatory bone destruction.  

PubMed

Pathological alterations in the balance of bone metabolism are central to the progression of inflammatory bone diseases such as periodontal disease. We have developed and characterized a novel ex vivo murine mandible model of inflammatory bone destruction. Slices of mandible were cultured for 14 days in the presence or absence of P. gingivalis lipopolysaccharide (LPS) or pro-inflammatory cytokines. Following culture, cell viability and tissue histomorphometry were assessed with quantification of matrix proteins, resident osteoclasts, ligament cells, monocytes, macrophages, and neutrophils. In the absence of inflammatory factors, culture viability, osteoclasts, and matrix components were maintained. LPS or TNF? stimulation demonstrated an increase in cellular proliferation, monocyte cells, osteoclast differentiation, and matrix degradation. Pathophysiological bone metabolism can be induced via exposure to LPS and direct influence of TNF? within the model despite the absence of systemic circulation, providing a model for inflammatory bone destruction and investigation of the effects of novel therapeutics. PMID:23857868

Sloan, A J; Taylor, S Y; Smith, E L; Roberts, J L; Chen, L; Wei, X Q; Waddington, R J

2013-08-01

111

Specific effect of immunomodulatory quinoline-3-carboxamide ABR-215757 in GM-CSF stimulated bone marrow cell cultures: block of initiation of proliferation of Gr-1+ cells.  

PubMed

Quinoline-3-carboxamides are currently in clinical development for treatment of both autoimmune disease and cancer. Carboxamides such as ABR-215757 (5757) have shown efficacy in several in vivo mouse models of human inflammatory autoimmune disease. Some microbial infections in mice cause GM-CSF dependent accumulation of dendritic cells expressing TNF? and inducible nitric oxide synthase (iNOS; Tip-DCs) in lymphoid organs. Functionally similar DCs develop in GM-CSF stimulated bone marrow (BM) cell cultures and offered an in vitro model that allowed us to study the impact of 5757 on cellular development of relevance for in vivo inflammatory conditions. We show in here that addition of 5757 to such cultures, in a dose-dependent way increased the frequency of DCs, while it reduced the frequency of Gr-1(+) cells by inhibiting their proliferation. This effect was specific as the compound neither influenced DC development from myeloid progenitors, nor the development of granulocytes in G-CSF stimulated BM cell cultures. Importantly, we also show that 5757 treatment reduced the accumulation of Gr-1(+) cells during inflammation in vivo. We therefore propose that this compound may ameliorate autoimmune disease by blocking proliferation of Gr-1(+) cells during inflammation-induced mobilization of myeloid cells. PMID:21388612

Helmersson, Sofia; Stenström, Martin; Leanderson, Tomas; Ivars, Fredrik

2011-08-01

112

Cadmium-Induced Bone Loss: Effects in Ovariectomized Mice and Osteoclast-Like Cells in Culture.  

National Technical Information Service (NTIS)

The research reported here was conducted to investigate the possibility that cadmium might be a factor that increases bone loss after the menopause. In our first study, we exposed female CF1 mice to a purified diet containing CdCl sub 2 at either 0.25, 5....

C. Kuhn D. P. Peterson E. S. Moretti M. H. Bhattacharyya T. M. Seed

1988-01-01

113

Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model.  

PubMed

Bone marrow-derived mesenchymal stem cells have become an attractive cell source for periodontal ligament regeneration treatment because of their potential to engraft to several tissue types after injury. Most researchers have focused on the transplantation process, but few have paid attention to cell safety concerns and rapid proliferation before transplantation. Using serum-free medium to culture stem cells may be an effective method to avoid problems associated with exogenous serum and the addition of growth factors to promote cell proliferation. Here, we randomly divided our serum-free cultures and treated them with different levels of epidermal growth factor (EGF). We then evaluated changes in rates of cell adhesion, proliferation, apoptosis, and cell cycle ratio as well as their differentiation potential. The data showed that all of these parameters were significantly different when comparing serum-free cultures with and without 10 nM/L EGF (p < 0.05/0.01); however, cells with 10 nM/L EGF did not respond differently than cells grown in standard serum-containing media without EGF (p > 0.05). In summary, our results demonstrate that 10 nM/L EGF was the optimal dose for serum-free culture, which can replace traditional standard serum medium for in vitro expansion of miniature pig bone marrow-derived mesenchymal stem cells. PMID:24002665

Wang, Xuan; Zheng, Feng; Liu, Ousheng; Zheng, Shutao; Liu, Yishan; Wang, Yuehong; Tang, Zhangui; Zhong, Liangjun

2013-12-01

114

Generation of large numbers of highly purified dendritic cells from bone marrow progenitor cells after co-culture with syngeneic murine splenocytes  

PubMed Central

Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes. From a total of 20×106 BM cells and 120×106splenocytes, 3 × 106 BM cells along with 20×106splenocytes were co-cultured in petri dishes for DC generation; 120×106 splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. The control group were BM cells cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8?, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA. The number of DCs derived from co-culture of BM and splenocytes (DCsTME) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2 % and 77.2%, respectively; p<0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCsTME compared to BM-derived DCs in the absence of splenocytes (193 pg/ml and 630 pg/ml, respectively; p<0.05). The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCsTME could be more advantageous for DC-based immunotherapy and vaccination techniques.

Kalantari, Tahereh; Kamali-Sarvestani, Eskandar; Zhang, Guang-Xian; Safavi, Farinaz; Lauretti, Elisabetta; Khedmati, Mohammad-Esmaeil; Rostami, Abdolmohamad

2013-01-01

115

Isolation of Rickettsia rickettsi in primary bone marrow cell and circulating monocyte cultures derived from experimentally infected guinea pigs.  

PubMed

Rickettsia rickettsi was isolated and propagated in primary cell cultures derived from experimentally infected guinea pigs. Organisms were recognized as early as 3 days after cultures were initiated. PMID:4197749

Buhles, W C; Huxsoll, D L; Elisberg, B L

1973-06-01

116

Isolation of Rickettsia rickettsi in Primary Bone Marrow Cell and Circulating Monocyte Cultures Derived from Experimentally Infected Guinea Pigs  

PubMed Central

Rickettsia rickettsi was isolated and propagated in primary cell cultures derived from experimentally infected guinea pigs. Organisms were recognized as early as 3 days after cultures were initiated. Images

Buhles, William C.; Huxsoll, David L.; Elisberg, Bennett L.

1973-01-01

117

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

SciTech Connect

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

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

1988-01-01

118

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

Microsoft Academic Search

The research reported here was conducted to investigate the possibility that cadmium might be a factor that increases bone loss after the menopause. In our first study, we exposed female CF1 mice to a purified diet containing CdCl2 at either 0.25, 5.0, or 50 ppM Cd starting at 70 days of age. After 12 months of exposure, mice were ovariectomized

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

1988-01-01

119

Human bone marrow mesenchymal stem cells support the derivation and propagation of human induced pluripotent stem cells in culture.  

PubMed

Human induced pluripotent stem cells (hiPSCs) need to be generated and expanded under clinically applicable culture conditions before they can be used for clinical application. In this study, we demonstrate that inactivated human mesenchymal stem cells (hMSCs) from different donors can be used as feeder cells to support the establishment and maintenance of hiPSCs. The hiPSCs we generated and expanded on hMSCs exhibited the typical morphology of human embryonic stem cells (hESCs), expressed undifferentiated pluripotent cell markers and genes, differentiated into all three germ layers via embryoid body and teratoma formation, and retained a normal chromosomal karyotype after 14 passages. However, we found that the rate of hiPSCs generation on hMSCs was 7.26%±2.09% compared with that on mouse embryonic fibroblasts (MEFs), and the calculated expansion efficiency of hiPSCs on hMSCs was lower than that on MEFs. hMSCs from various donors and different passages did not influence the results. These findings suggest that hMSCs can be used as feeder cells to derive and maintain hiPSCs, and thus provide another clinically feasible method for generating and expanding hiPSCs. However, the cytokines and adhesion molecules in this system should be identified to develop a preferable clinical culture condition for hiPSCs. PMID:23713432

Zhang, Lifei; Zheng, Weiyan; Wang, Yebo; Wang, Yingjia; Huang, He

2013-06-01

120

Regulation of early hematopoiesis in serum-deprived cultures of mafosfamide-treated and untreated CD34-enriched bone marrow cells.  

PubMed

Our experiments have addressed the regulation of early hematopoietic progenitor cell expansion by interleukin 3 (IL-3) and interleukin 1 beta (IL-1 beta) and its modulation by bone marrow fibroblasts in vitro. In a two-stage assay utilizing serum-deprived (SD) presuspension cultures of CD34-enriched bone marrow (BM) cells followed by clonal cultures, absolute numbers of granulocyte-macrophage progenitor cells (day-14 granulocyte-macrophage colony-forming units [CFU-GM]) increased progressively to 164% and 204% of input levels after 12 days of culture in the presence of IL-3 alone or in combination with IL-1 beta, respectively. Multilineage (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) and erythroid (erythroid burst-forming units, BFU-E) progenitor cell numbers increased above or were maintained at input levels after 4 and 7 days of liquid culture in the presence of IL-3 and IL-3 plus IL-1 beta, respectively, but in contrast to granulocyte-macrophage colony-forming units (CFU-GM) they were essentially undetectable after 12 days of culture. Progenitors more primitive than colony-forming cells (pre-CFU) were assessed in SD-presuspension cultures of CD34-enriched BM cells purged with mafosfamide to eliminate base-line CFU-GM, CFU-GEMM, and BFU-E. Under these conditions and in the absence of stromal elements, CFU-GM but neither CFU-GEMM nor BFU-E developed in response to cytokines alone. In the additional presence of passaged bone marrow fibroblasts, however, IL-3 plus IL-1 beta and to a lesser degree IL-3 alone induced a pronounced amplification of BFU-E and CFU-GEMM, indicating that their development from a more primitive progenitor compartment requires growth activities in addition to IL-3 and IL-1 beta that are provided by marrow-derived stromal cells such as fibroblasts. PMID:1714403

Ottmann, O G; Stella, C C; Eder, M; Reutzel, P; Ströcker, S; Hoelzer, D; Ganser, A

1991-09-01

121

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

PubMed

Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the consequent bone formation in vivo were significantly enhanced. These results suggest that the perfusion culture system is a valuable and convenient tool for applications in tissue engineering, especially in the generation of artificial bone tissue. PMID:14670108

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

2003-12-01

122

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture  

PubMed Central

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ? 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.

Choi, Mi Ran; In, Yong-Ho; Park, Jungsun; Park, Taesung; Jung, Kyoung Hwa; Chai, Jin Choul; Chung, Mi Kyung; Lee, Young Seek

2012-01-01

123

Differentiation of Hematopoietic Cells in Long-Term Bone Marrow Culture.  

National Technical Information Service (NTIS)

The development of in vitro clonal assay techniques for hematopoietic cells in 1965-1966 has provided invaluable information on the characteristics of progenitor cells restricted to specific lineages. These techniques have now been adapted to yield coloni...

A. J. Salvado R. C. Meagher D. G. Wright

1984-01-01

124

Therapeutic angiogenesis by autologous bone marrow cell implantation together with allogeneic cultured dermal substitute for intractable ulcers in critical limb ischaemia.  

PubMed

Therapeutic angiogenesis by autologous bone marrow cell implantation improves blood supply in patients with critical limb ischaemia. In addition, allogeneic cultured dermal substitute is effective for intractable ulcers. The present study determined the effectiveness of bone marrow cell implantation combined with allogeneic cultured dermal substitute in treating severely ischaemic ulcers. We treated eight consecutive patients with severely ischaemic ulcers using this procedure. Stromal cells aspirated from bone marrow were processed to obtain suspensions of mononuclear cells, platelets and endothelial progenitor cells and immediately injected intramuscularly into the lower leg and around the wound, on which allogeneic cultured dermal substitute was applied and changed weekly. Skin ulcers were subsequently closed by skin grafting, if necessary. Angiogenesis was confirmed by postoperative analyses such as ankle-brachial pressure index, angiography, thermography and (99m)Technetium-Tetrofosmin perfusion scintigraphy. Above- or below-knee amputation was avoided in all patients and wounds were completely closed in six of them. These results indicate that this combined therapy effectively treated ischaemic ulcers. Since the incidence of this condition might increase in the future, this therapeutic approach should play an important role in the preservation of ischaemic limbs. PMID:20060793

Mizuno, Hiroshi; Miyamoto, Masaaki; Shimamoto, Minoru; Koike, Sachiko; Hyakusoku, Hiko; Kuroyanagi, Yoshimitsu

2010-11-01

125

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

PubMed Central

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

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

2004-01-01

126

Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture  

Microsoft Academic Search

Until now, there has been no in vitro model that duplicates the environment of bone marrow. The purpose of this study was to analyze proliferation and differentiation of human bone marrow stromal cells (hBMSC) under the influence of continuous perfusion and cyclic mechanical loading.hBMSC of seven individuals were harvested, grown in vitro, and combined. 106 hBMSC were seeded on a

M. Jagodzinski; A. Breitbart; M. Wehmeier; E. Hesse; C. Haasper; C. Krettek; J. Zeichen; S. Hankemeier

2008-01-01

127

Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts  

Microsoft Academic Search

In the traditional Korean medicine, Drynariae Rhizoma (DR) [Drynaria fortunei (kunze) J. Sm] has been reported as a good enhancer for bone healing. In this experiment, we investigate the effects of DR on bone resorption using the bone cells culture. Different concentrations of crude extract of DR were added to mouse bone cells culture. The mitochondria activity of the bone

Ji-Cheon Jeong; Sung-Koo Kang; Cheol-Ho Youn; Chang-Whan Jeong; Hyung-Min Kim; Young-Choon Lee; Young-Chae Chang; Cheorl-Ho Kim

2003-01-01

128

A comprehensive study on optimization of proliferation and differentiation potency of bone marrow derived mesenchymal stem cells under prolonged culture condition.  

PubMed

Bone marrow derived stem cells (BMSC) have paved way to clinical approaches for its utilization in a variety of diseases due to its ease of isolation combined with its multilineage differentiation capacity. However, the applicability of BMSC is not successful due to the lesser number of nucleated cells obtained from large samples. Hence, culture expansion of BMSC is a prerequisite, as high numbers of stem cells are needed to meet the standards of clinical advancement. There are attempts on optimizing culture condition for large scale production of BMSC. It was believed that, prolonged culture of BMSC is difficult since they tend to lose their characteristics and differentiation potential. Hence, our study aims to determine whether BMSCs could retain its proliferative and differentiation capacity in prolonged in vitro culture by a comparative study on extensive culturing of BMSC with the following four media, DMEM LG (DMEM-Low Glucose), DMEM KO (DMEM-Knock Out), Alpha MEM (Alpha Minimal Essential Medium), DMEM F 12. We found that two samples among the three cultured tend to lose their property in long term culturing. Besides, we also found that DMEM LG and Alpha MEM were the optimal media for in vitro culturing of BMSC. Overall, it was concluded that BMSC can be cultured until passage 15 without losing its characteristics. However, its potency beyond passage 15 has to be further elucidated for utilization of the ex vivo expanded BMSC for subsequent cellular therapies. PMID:22729554

Dhanasekaran, M; Indumathi, S; Lissa, R P; Harikrishnan, R; Rajkumar, J S; Sudarsanam, D

2013-03-01

129

Increased production of IFN-? by natural killer cells triggered with bone marrow-derived dendritic cells cultured in the presence of retinoic acid.  

PubMed

All-trans-retinoic acid (RA), a vitamin A metabolite, is beginning to be explored as an immunopharmacologic agent. While its effects on dendritic cells and the induction of regulatory T cells have been recognized, little is known about the effect of RA on dendritic cell-natural killer cell (DC-NK) crosstalk. DC-NK crosstalk is important in directing the innate immune response, as well as subsequent adaptive immune response during viral infection, cancer, pregnancy, as well as organ transplantation. Here we demonstrate with flow cytometry and cytokine bead array analysis, that bone marrow derived dendritic cells (BMDCs) cultured in the presence of ?98% HPLC purified RA (RA-DCs) were suppressed in their ability to mature in response to TLR stimulation. 1 µM of RA was found to be optimal without affecting the percentage of DCs in culture. Upregulation of MHCII and costimulatory molecule CD86, as well as IL-12 secretion were inhibited by RA treatment. RA-DCs differentially modulate NK cell function compared to BMDCs. In vitro co-culture of RA-DCs with NK cells reveal increased IFN-? secretion. Increased production of IFN-? in lung NK cells was also demonstrated when RA-DCs were injected into the tail vein. Our results suggest that RA-DCs exhibit a regulatory phenotype and function, which differentially modulates NK cell function. Furthermore, IFN-? has various regulatory and immunological functions, depending on the immunological context. The effect of RA-DCs needs to be further explored in the context of a disease in order to understand the regulatory effects of retinoic acid. PMID:23701916

Chau, Jessie; Moza, Dasha; Hossain, Nazia; Lee, Jeffrey K; Bienenstock, John; Karimi, Khalil

2013-09-01

130

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

SciTech Connect

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

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

1982-07-01

131

Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4.  

PubMed

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis. The CRPV model has been used to test various preventative and therapeutic vaccination strategies, and the availability of rabbit DCs would further expand its utility. However, to date, rabbit DCs have not been phenotypically and/or functionally characterized. Here we show that DCs can be generated in vitro from rabbit bone marrow mononuclear cells (BMMCs) cultured in the presence of the human cytokines GM-CSF and IL-4 and matured with lipopolysaccharide (LPS). These cells show upregulation of MHC class II and CD86, as well as downregulation of CD14, do not have non-specific esterase activity, are able to perform receptor-mediated endocytosis, and are potent stimulators of allogeneic T cell proliferation in mixed lymphocyte reactions. The ability to generate rabbit DCs makes it possible to test the efficacy of DC vaccination in the prevention and treatment of CRPV-induced lesions, which may provide useful preclinical data regarding the use of DC vaccines for HPV-associated lesions, including cervical cancer. PMID:15621303

Cody, Virginia; Shen, Hong; Shlyankevich, Mark; Tigelaar, Robert E; Brandsma, Janet L; Hanlon, Douglas J

2005-02-10

132

Bone Reconstruction with Bone Marrow Stromal Cells  

Microsoft Academic Search

Bone marrow stromal\\/stem cells (BMSCs) are multipotent adult stem cells and have become the important cell source for cell therapy and engineered tissue repair. Their osteogenic differentiation potential has been well characterized in many in vitro studies. In addition, small animal model–based studies also reveal their capability of bone formation in vivo when implanted with biodegradable scaffold, indicating the great

Wei Liu; Lei Cui; Yilin Cao

2006-01-01

133

Voltage-dependent potentiation of low-voltage-activated Ca2+ channel currents in cultured rat bone marrow cells.  

PubMed Central

1. The whole-cell patch-clamp technique was used to study Ca2+ channel currents in stromal cells of 7-10 day dexamethasone-treated and control rat bone marrow cultures. In saline containing either 108 mM Ba2+ or a 2.5 mM Ca(2+)-1 mM Mg2+ mixture, most cells expressed both fast-inactivating, low-voltage-activated (LVA) and slow-inactivating, high-voltage-activated (HVA) currents. 2. Repeated application of 400 ms voltage steps to 60 mV above the holding potential (Vh, -90 mV in Ca(2+)-Mg2+ mixture and -60 mV in Ba2+) at a frequency > or = 0.1 Hz resulted in a potentiation of the LVA component of the 2nd and subsequent currents. 3. LVA current potentiation was examined using a two-pulse (prepulse-test pulse) method. Prepulses to Vh + 150 mV induced an 80-100% increase in the amplitude of the LVA component of Ca2+ channel currents in saline containing either Ba2+ or Ca(2+)-Mg2+. This effect was also seen in non-dexamethasone-treated cultures. 4. Potentiation was not modified by omission of ATP and GTP from the pipette saline, and was not inhibited by extracellular application of the broad spectrum kinase inhibitors H-7 or RK252-a. 5. Potentiation was dependent on the amplitude and duration of the prepulse. Using the standard protocol, the threshold for potentiation was approximately Vh + 45 mV and saturation occurred at Vh + 150-180 mV. Further increases in prepulse amplitude did not modify potentiation. With a prepulse to +10 mV (Ba2+ saline) potentiation was half-maximal with a prepulse duration of 250-300 ms duration and saturated at 750-1000 ms. 6. Peak potentiation occurred 1-2 s after the prepulse. The time for total decay of potentiation varied from 10 to 90 s. 7. Voltage dependency of prepulse-induced potentiation did not resemble that of inactivation induced by similar prepulses. 8. Current kinetics, I-V relationship and sensitivity to blockade by Ni2+ and diphenylhydantoin of prepulse-recruited current resembled those of control LVA current. 9. The amplitude of prepulse-recruited current was positively correlated with control LVA current amplitude. 10. LVA currents supported regenerative potentials under current clamp. Repeated activation reduced spike latency. 11. It is suggested that current potentiation may be recruited physiologically, possibly in association with activation of stretch-sensitive channels, causing enhanced activation of HVA Ca2+ currents.

Publicover, S J; Preston, M R; El Haj, A J

1995-01-01

134

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

PubMed

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

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

2008-08-01

135

Human amniotic fluid stem cells culture onto titanium screws: a new perspective for bone engineering.  

PubMed

The use of titanium plates and screws for osteosynthesis is considered to be an effective treatment for different kinds of fractures in orthopedic surgery. The aim of the present study is to test the ability of titanium screws to promote the growth of osteoblasts obtained from human amniotic fluid stem cells (AFS). Osteoblastic differentiation was assessed by RT-PCR of specific markers such as COL1, ONC, OPN, OCN, OPG, BMP-4 and Runx2. Mineralization was demonstrated by the presence of red depositions. Adherent cells were found to cover the whole surface of titanium screw by Scanning Electron Microscopy (SEM). The result indicates the excellent growth of osteoblasts obtained from amniotic fluid on a titanium surface and could represent an important point in view of a possible therapeutic application of AFS cells. PMID:20003768

Antonucci, I; Pantalone, A; De Amicis, D; D'Onofrio, S; Stuppia, L; Palka, G; Salini, V

2009-01-01

136

Separation of Spleen Colony Forming Units (CFU-S) from Mouse Bone Marrow Cells Using Velocity Sedimentation in an Isokinetic Gradient of Ficoll in Tissue Culture Medium  

PubMed Central

Spleen colony forming cells (CFU-S) from mouse bone marrow were concentrated using velocity sedimentation in an isokinetic gradient of Ficoll (polysucrose) in tissue culture medium. Following separation, CFU-S were found as a sharp modal population of cells which was discrete from the modal populations of lymphocytes and other cell types. Despite the wide separation of lymphocytes, granulocytes and CFU-S in the density gradient, there was no appreciable resolution of CFU-S specifically committed to erythroid, granulocytic or undifferentiated colony formation. This suggests that committed stem cells either share identical physical properties or are not detected by the spleen colony assay. In the purest fractions from the isokinetic gradient, CFU-S were 2.8- to 4.7-fold purified when compared with mouse bone marrow before separation. ImagesFig 1Fig 2Fig 3

Pretlow, Thomas G.; Williams, Edwin E.; Davis, Maxie L.; Zettergren, Judy G.

1973-01-01

137

Effect of cadmium on bone resorption in cultured fetal bone  

SciTech Connect

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

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

1980-08-01

138

Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.  

PubMed

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

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

2014-03-01

139

Effect of Lithium Chloride on Proliferation and Bone Differentiation of Rat Marrow-Derived Mesenchymal Stem Cells in Culture  

Microsoft Academic Search

Objective(s) It is believed that the mesenchymal stem cell (MSC) differentiation and proliferation are the results of activation of wnt signaling pathway. On the other hand, lithium chloride is reported to be able to activate this pathway. The objective of this study was to investigate the effect of lithium on in vitro proliferation and bone differentiation of marrow-derived MSC. Materials

Mohamadreza Baghaban Eslaminejad; Mahmood Talkhabi; Bahman Zeynali

2008-01-01

140

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

PubMed

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine. PMID:22304383

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

2012-10-01

141

Serum Free Cultured Bone Marrow Mesenchymal Stem Cells as a Platform to Characterize the Effects of Specific Molecules  

Microsoft Academic Search

Human mesenchymal stem cells (hMSC) are easily isolated from the bone marrow by adherence to plastic surfaces. These cells show self-renewal capacity and multipotency. A unique feature of hMSC is their capacity to survive without serum. Under this condition hMSC neither proliferate nor differentiate but maintain their biological properties unaffected. Therefore, this should be a perfect platform to study the

Leonardo Solmesky; Sharon Lefler; Jasmine Jacob-Hirsch; Shlomo Bulvik; Gideon Rechavi; Miguel Weil

2010-01-01

142

Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material, consisting of poly-L-lactide, carbon nanotubes and microhydroxyapatite, in the presence of bone morphogenetic protein-2.  

PubMed

In bone tissue engineering composite materials have been introduced, combining a degradable polymer matrix with, for instance, carbon nanotubes (CNTs) to improve mechanical properties or with microhydroxyapatite (?HA) to improve osteoconduction. The addition of bone morphogenetic protein-2 (BMP-2) can further improve the biological response to the material. However, the influence of such an elaborate composite formation on osteoprogenitor cells is unknown. To examine this, rat bone marrow (RBM) cells were cultured on porous poly-L-lactic acid and composite scaffolds, with or without added BMP-2. Cell proliferation and differentiation were studied using DNA, alkaline phosphatase and scanning electron microscopic analysis. Further, genetic profiles were examined by microarray investigation. Results showed that the composite scaffold had no significant effect on the proliferation of RBM cells, but indicated a negative effect on cell differentiation. The addition of BMP-2 also had no significant effect on the proliferation of RBM cells, but differentiation towards the osteogenic lineage was confirmed. In the arrays results, the addition of BMP-2 alone led to the expression of genes involved in (minor) inflammation. The composite scaffold, and even more distinctly the combination of the composite scaffold with BMP-2, led to the expression of genes, based on gene ontology, connected to tumorigenesis. Therefore, CNT- and ?HA-containing composite materials are not recommended as a bone restorative material. PMID:20601234

van der Zande, Meike; Walboomers, X Frank; Brännvall, Mathias; Olalde, Beatriz; Jurado, Maria J; Alava, J Iñaki; Jansen, John A

2010-11-01

143

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

PubMed Central

Background 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. Methods 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. Results 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. Conclusions 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.

2013-01-01

144

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

SciTech Connect

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

Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China)] [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China)] [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong)] [Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Xiao, Zhi-Cheng, E-mail: zhicheng.xiao@monash.edu [Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical College, Kunming 650031 (China) [Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical College, Kunming 650031 (China); Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Melbourne 3800 (Australia)

2012-05-25

145

Retinoic acid induces mouse bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) stem cells into male germ-like cells in a two-dimensional cell culture system.  

PubMed

We have examined the effect of retinoic acid (RA) on differentiation of bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells. Bone marrow stem cells (BMSCs) were isolated from the femur of 3-4-week-old male C57BL/6 mice. Magnetic-activated cell sorting (MACS) system was used to sort CD15(+) , Oct4(+) and CXCR4(+) cells. RT-PCR was used to follow the expression of pluripotency markers. Sorted CD15(+) , Oct4(+) and CXCR4(+) cells were cultured in an undifferentiated condition on a feeder layer of mitomycin C-inactivated C2C12. The embryoid-like bodies were differentiated into male germ cells by retinoic acid. To identify the expression of male germ specific markers, differentiated cells were analysed by means of reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence staining. RT-PCR and immunofluorescence show that bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells express pluripotency markers, Oct4, Nanog, Rex-1, SOX-2 and AP. The purified CD15(+) , Oct4(+) and CXCR4(+) formed structures like embryoid bodies when plated over a feeder layer; these bodies were alkaline phosphatase positive. When cells were induced by RA, bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) were positive for Mvh, Dazl, Piwil2, Dppa3 and Stra8, that known molecular markers of male germ cells. Thus RA can induce differentiation of mouse bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells in vitro. Negative results for the gene expression analysis of female germ cells markers, GDF9 and ZP3, confirmed this conclusion. PMID:24677291

Kashani, Iraj Ragerdi; Zarnani, Amir Hassan; Soleimani, Masoud; Abdolvahabi, Mir Abbas; Nayernia, Karim; Shirazi, Reza

2014-06-01

146

Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system.  

PubMed

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrix scaffold. However, the eventual practical application of BMSCs in such 3D scaffolds is limited by the low seeding efficiency of the cells within the scaffold. Here, we demonstrated that the cell seeding efficiency of BMSCs in the Cytomatrix scaffold can be improved significantly (t-test, p<0.05) by means of macroencapsulating the scaffold via the complex coacervation of a methylated collagen and terpolymer. The thickness and density of the polyeletrolyte complex can be modulated by the contact time between the methylated collagen and terpolymer to balance between cell entrapment efficacy and mass transfer impedance imparted by the complex. Porcine BMSCs were macroencapsulated in Cytomatrix scaffolds using various polyelectrolyte contact time and cultured under both static and dynamic conditions. Throughout the range of contact time investigated, macroencapsulation did not affect the viability of the porcine BMSCs in dynamic culture. However, the viability of the cells under static cultures was compromised with longer polyelectrolyte contact time. Therefore, this proposed method of macroencapsulation enables customization to achieve enhanced seeding efficiency without mass transfer impedance for different culture configurations. PMID:15664642

Toh, Yi-Chin; Ho, Saey Tuan; Zhou, Yi; Hutmacher, Dietmar W; Yu, Hanry

2005-07-01

147

Phenotypic and cytologic studies of lymphoid cells and monocytes in primary culture of porcine bone marrow during infection of African swine fever virus.  

PubMed

We have modeled in vitro infection of African swine fever virus (ASFV) in primary unstimulated cells of the porcine bone marrow and have studied the phenotypical changes in the population of porcine lymphoid cells by cytophotometry. Monocytes and large-sized lymphocytes completely vanished in 72 h of infection which is result of high sensitivity of those cells to ASFV. We describe DNA synthesis in monocytes at 24 h post infection. Cytophotometry of the uninfected cells revealed the few number of atypical lymphocytes and lymphoblasts after 72 h of cultivation; whereas in viral infected cultures, atypical cells appeared in large quantity (about 14%) with 24 h. Most of atypical lymphocytes and lymphoblasts had altered nucleus, and only a small number of atypical cells had additional nucleus. The cytophotometry of main and additional nuclei showed that DNA content didn't exceed diploid standard which indicates that the additional nuclei were consequence of fragmentation of nuclei in lymphocytes. PMID:21184199

Karalova, E M; Sargsyan, Kh V; Hampikian, G K; Voskanyan, H E; Abroyan, L O; Avetisyan, A S; Hakobyan, L A; Arzumanyan, H H; Zakaryan, H S; Karalyan, Zaven A

2011-03-01

148

Combination of enzymes and flow perfusion conditions improves osteogenic differentiation of bone marrow stromal cells cultured upon starch/poly(epsilon-caprolactone) fiber meshes.  

PubMed

Previous studies have shown that alpha-amylase and lipase are capable of enhancing the degradation of fiber meshes blends of starch and poly(epsilon-caprolactone) (SPCL) under dynamic conditions, and consequently to promote the proliferation and osteogenic differentiation of bone marrow stromal cells (MSCs). This study investigated the effect of flow perfusion bioreactor culture in combination with enzymes on the osteogenic differentiation of MSCs. SPCL fiber meshes were seeded with MSCs and cultured with osteogenic medium supplemented with alpha-amylase, lipase, or a combination of the two for 8 or 16 days using static or flow conditions. Lipase and its combination with alpha-amylase enhanced cell proliferation after 16 days. In addition, the flow perfusion culture enhanced the infiltration of cells and facilitated greater distribution of extracellular matrix (ECM) throughout the scaffolds in the presence/absence of enzymes. A significant amount of calcium was detected after 16 days in all groups cultured in flow conditions compared with static cultures. Nevertheless, when alpha-amylase and lipase were included in the flow perfusion cultures, the calcium content was 379 +/- 30 microg/scaffold after as few as 8 days. The highest calcium content (1271 +/- 32 microg/scaffold) was obtained for SPCL/cell constructs cultured for 16 days in the presence of lipase and flow. Furthermore, von Kossa staining and tetracycline fluorescence of histological sections demonstrated mineral deposition within the scaffolds for all groups cultured for 16 days under flow. However, all the data corroborate that lipase coupled with flow perfusion conditions improve the osteogenic differentiation of MSCs and enhance ECM mineralization. PMID:20694973

Martins, Ana M; Saraf, Anita; Sousa, Rui A; Alves, Catarina M; Mikos, Antonios G; Kasper, F Kurtis; Reis, Rui L

2010-09-15

149

An ex vivo rodent mandible culture model for bone repair.  

PubMed

To understand fully cellular mechanisms during bone tissue repair and engineering, there is a need to develop reproducible three-dimensional organotypic culture models, whereby cells in their natural extracellular matrix can be manipulated. Limitations in current model systems do not allow for this integrated approach. This study aimed to develop and validate an ex vivo fractured rat mandible model, to investigate specific molecular and cellular processes involved in bone repair. Slices of mandible from 28-day-old male Wistar rats were cultured in Trowel-type cultures at the liquid-gas interface for up to 21 days. Maintenance of cell and tissue architecture and viability was shown within fractured mandible slices during all culture periods. Autoradiographic studies demonstrated that resident cells were actively synthesizing and secreting proteins, and cells of the osteoblast lineage were shown to survive throughout the culture periods. The model was responsive to exogenously added transforming growth factor-?1, with observed increases in cellular migration/proliferation and expression of bone matrix proteins. The ex vivo mandible model developed within this study may represent an ideal system for investigating specific processes of bone repair, as well as a promising alternative to in vivo testing of novel clinical therapeutics. PMID:20218818

Smith, Emma L; Locke, Matthew; Waddington, Rachel J; Sloan, Alastair J

2010-12-01

150

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

NSDL National Science Digital Library

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

Carl A. Gregory (Tulane University Health Sciences Center;Center for Gene Therapy REV); Joni Ylostalo (Tulane University Health Sciences Center;Center for Gene Therapy REV); Darwin J. Prockop (Tulane University Health Sciences Center;Center for Gene Therapy REV)

2005-07-26

151

Osteoblast-like Differentiation of Cultured Human Coronary Artery Smooth Muscle Cells by Bone Morphogenetic Protein Endothelial Cell Precursor-derived Regulator (BMPER)*  

PubMed Central

Differentiation of vascular smooth muscle cells (SMCs) into osteoblast-like cells is considered to be a mechanism of vascular calcification. However, regulators of osteoblast-like differentiation of vascular SMCs are not fully elucidated. Here, we investigated the expression of bone morphogenetic protein (BMP)-binding endothelial cell precursor-derived regulator (BMPER), a vertebrate homologue of Drosophila crossveinless-2, in vascular SMCs and the role and mode of action of BMPER in osteoblast-like differentiation of human coronary artery SMCs (HCASMCs). BMPER was expressed in cultured human vascular SMCs, including HCASMCs. Silencing of endogenous BMPER expression by an RNA interference technique inhibited osteoblast-like differentiation of HCASMCs, as evaluated by up-regulation of osteoblast markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), by down-regulation of a SMC marker ?-smooth muscle actin (?SMA), and by mineralization. Treatment with recombinant BMPER enhanced, whereas BMP-2 reduced osteoblast-like differentiation. BMPER antagonized BMP-2-induced phosphorylation of Smad 1/5/8, suggesting that the effect of BMPER was mediated by antagonizing the action of BMP. BMPER increased I?B? phosphorylation and NF-?B activity and specific NF-?B decoy oligonucleotides deteriorated osteoblast-like differentiation of HCASMCs by BMPER. In human coronary artery with atherosclerotic plaque containing calcification, the BMPER-positive signals were observed in the neointimal and medial SMCs in the vicinity of the plaque. These findings indicate that BMPER is a novel regulator of the osteoblast-like differentiation of HCASMCs.

Satomi-Kobayashi, Seimi; Kinugasa, Mitsuo; Kobayashi, Reiko; Hatakeyama, Kinta; Kurogane, Yusuke; Ishida, Tatsuro; Emoto, Noriaki; Asada, Yujiro; Takai, Yoshimi; Hirata, Ken-ichi; Rikitake, Yoshiyuki

2012-01-01

152

Human liquid bone marrow culture in serum-free medium.  

PubMed

Prolonged in vitro maintenance of human bone marrow progenitor cells was achieved using a serum-free (SF) liquid culture system. Culture medium was based on Iscove's medium supplemented with bovine serum albumin, human transferrin, bovine insulin, soybean lecithin, cholesterol, hydrocortisone and alpha-thioglycerol. Under these standardized culture conditions, CFU-GM were maintained for up to 4 weeks, as is the case when using conventional serum-dependent medium. Erythropoiesis exhibited a slower decline than that found using serum containing medium. Development of normal haematopoiesis was effective in spite of poor stromal cell development--a confluent adherent layer as classically described in serum conditions was never achieved. Our newly defined system provides a reliable technique for studying human haematopoietic stem cell proliferation and differentiation in vitro; it allows for rational utilization of currently available purified recombinant growth factors. It may be a promising tool in the clinical use of cultured haematopoietic stem cells. PMID:2818936

Drouet, X; Douay, L; Giarratana, M C; Baillou, C; Gorin, N C; Salmon, C; Najman, A

1989-10-01

153

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: Light and electron microscopic study  

Microsoft Academic Search

Summary The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension

Junya Kanehisa; Toshiyuki Izumo; Mikio Takeuchi; Takeshi Yamanaka; Teruhisa Fujii; Hiroshi Takeuchi

1991-01-01

154

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films.  

PubMed

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-? and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation. PMID:24840613

Baker, Bryan A; Pine, P Scott; Chatterjee, Kaushik; Kumar, Girish; Lin, Nancy J; McDaniel, Jennifer H; Salit, Marc L; Simon, Carl G

2014-08-01

155

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.  

PubMed Central

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression. Images

Chang, S M; Wager-Smith, K; Tsao, T Y; Henkel-Tigges, J; Vaishnav, S; Caskey, C T

1987-01-01

156

Biochemical properties and mechanism of action of a vanadyl(IV)-aspirin complex on bone cell lines in culture.  

PubMed

A recently synthesized vanadyl(IV) complex with aspirin [VO(aspirin)ClH2O]2, has been thoroughly investigated by physicochemical techniques. In order to support the proposed structure, stoichiometry and the coordination sphere of the vanadium center, some studies such as elemental analysis, electronic (diffuse reflectance) and vibrational (infrared) spectroscopies, magnetic susceptibility, as well as the thermal behavior, were carried out. The bioactivity of the vanadium complex (VOAspi) was evaluated on two osteoblast-like cell lines in culture, being its cytotoxic effects stronger than the vanadyl cation as assessed by morphological changes and lipid peroxidation. These effects may be partially explained through the induction of the expression of Erks (Extracellular signal-regulated kinases) and the inhibition of the PTPases (Phosphotyrosine phosphatases) present in the cellular extracts. PMID:11865824

Etcheverry, Susana B; Williams, Patricia A M; Sálice, Viviana C; Barrio, Daniel A; Ferrer, Evelina G; Cortizo, Ana M

2002-03-01

157

Avian medullary bone in organ culture: Effects of vitamin D metabolites on collagen synthesis  

Microsoft Academic Search

Summary  A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence\\u000a of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of3H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial\\u000a collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The

John R. Harrison; Nancy B. Clark

1986-01-01

158

Eliminating the need of serum testing using low serum culture conditions for human bone marrow-derived mesenchymal stromal cell expansion  

PubMed Central

Background The conventional expansion of human mesenchymal stromal cells (hMSC) for tissue engineering or (pre-) clinical investigation includes the use of 10% fetal bovine serum (FBS). However, there exists immense lot-to-lot variability in FBS samples and time consuming as well as cost intensive lot pre-testing is essential to guarantee optimal hMSC proliferation and stem cells characteristics maintenance. Furthermore, lot-to-lot variability impedes the long-term consistency of research and comparability between research groups. Therefore, we investigated the use of defined, invariable, non-synthetic FBS in low serum culture conditions for isolation and expansion of hMSC. Methods hMSC were isolated from bone marrow in Panserin 401 supplemented with growth factors and 2% MSC-tested or non-tested, defined, invariable, non-synthetic FBS and further cultivated in vitro. The surface marker expression, differentiation capacity as well as cell proliferation and cytotoxicity was analyzed and compared between serum samples. Results Cells isolated and cultivated with low concentrations of MSC-tested or non-tested FBS demonstrated no differences in surface marker expression or differentiation capacity. Proliferation of hMSC was equal in medium supplemented with either serum with no indication of cell death. Conclusions The low serum concentration in Panserin 401 supplemented with growth factors enables the use of defined, invariable, non-synthetic FBS for the isolation and expansion of hMSC. The required hMSC characteristics like surface marker expression and differentiation capacity are maintained. Importantly, no differences in the cell proliferation could be detected. Therefore, using these low-serum culture conditions, the need for lot-to-lot pre-testing of FBS usually needed for optimal hMSC expansion is abolished leading to long-term consistency and comparability of results.

2013-01-01

159

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

PubMed Central

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

2014-01-01

160

The Clinical Use of Human Culture— Expanded Autologous Bone Marrow Mesenchymal Stem Cells Transplanted on Platelet-Rich Fibrin Glue in the Treatment of Articular Cartilage Defects: A Pilot Study and Preliminary Results  

Microsoft Academic Search

Objective: To test the hypothesis that platelet-rich fibrin glue (PR-FG) can be used clinically as a scaffold to deliver autologous culture-expanded bone marrow mesenchymal stem cells (BM-MSCs) for cartilage repair and to report clinical results 1 y after implantation of MSCs PR-FG. Patients and Methods: Autologous BM-MSCs were culture expanded, placed on PR-FG intraoperatively, and then transplanted into 5 full-thickness

Amgad M. Haleem; Abdel Aziz El Singergy; Dina Sabry; Hazem M. Atta; Laila A. Rashed; Constance R. Chu; Mohammed T. El Shewy; Akram Azzam; Mohammed T. Abdel Aziz

2010-01-01

161

Association of Oxidative Stress with Postmenopausal Osteoporosis and the Effects of Hydrogen Peroxide on Osteoclast Formation in Human Bone Marrow Cell Cultures  

Microsoft Academic Search

It has been suggested that oxidative stress is associated with the pathogenesis of osteoporosis. The objective of this study\\u000a was to explore the association between a marker of oxidative stress and either bone turnover markers or bone mineral density\\u000a (BMD) in postmenopausal women. In addition, the effects of oxidative stress on the formation of osteoclasts in human bone\\u000a marrow cell

Ki Hyun Baek; Ki Won Oh; Won Young Lee; Seong Su Lee; Mee Kyoung Kim; Hyuk Sang Kwon; Eun Jung Rhee; Je Ho Han; Ki Ho Song; Bong Yun Cha; Kwang Woo Lee; Moo Il Kang

2010-01-01

162

Co?culture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes.  

PubMed

Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy. This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs. PMID:24271232

Lehmann, Tomasz P; Filipiak, Krystyna; Juzwa, Wojciech; Sujka-Kordowska, Patrycja; Jagodzi?ski, Pawe? P; Zabel, Maciej; G?owacki, Jakub; Misterska, Ewa; Walczak, Micha?; G?owacki, Maciej

2014-02-01

163

"In-bone" utricle cultures - A simplified, atraumatic technique for in situ cultures of the adult mouse (Mus musculus) utricle  

PubMed Central

Hypothesis The “in-bone” method of culturing utricles described here is a reliable and atraumatic technique for culturing mature mouse hair cells and studying hair cell death and protection. Background The current in vitro technique for studying hair cells of the mature mouse utricle involves removal from the temporal bone and free floating culture in media. This technique can be problematic due to variability in the preservation of the sensory epithelium and a steep learning curve that results in injury of the sensory epithelium in less experienced hands. We present a new atraumatic technique of culturing the utricle in situ within the temporal bone. Methods Leaving the temporal bone largely intact, a window is opened in the bony vestibule overlying the mouse utricle. The entire temporal bone is then placed into culture media. Utricles were cultured in situ for several days with minimal damage to the epithelium. The utricles are then fixed in situ, removed from the temporal bone, and processed. A standardized aminoglycoside-induced hair cell damage protocol was developed. Results Mature mouse utricles maintained hair cell numbers for 3 days in culture. Exposure to neomycin resulted in significant dose-dependent hair cell toxicity (p<.0001, one-way ANOVA). Exposure to the protective drug tacrine resulted in significant protection against neomycin (p<.05, three-way ANOVA). Conclusion The “in-bone” technique is a reliable and atraumatic method for culturing mature mouse utricles and studying hair cell death and protection. It is easily mastered and can make in vitro study of hair cells accessible to more research groups.

Ou, Henry C.; Lin, Vincent; Rubel, Edwin W

2013-01-01

164

Bone marrow stem cells and biological scaffold for bone repair in aging and disease.  

PubMed

The loss of bone mass observed in aging enhances the risk of fractures. The process of bone repair in aging is slow and limited due to reduced activity of the osteoblasts. Bone marrow stem cells (MSCs) residing in the bone marrow are the progenitors for osteoblasts. The ability to enhance healing of bone defect in aging by MSCs can contribute in the prevention of the complications resulting from long-term immobilization that are especially fatal in old age. Our aim was to test the ability of MSCs inserted into a biological scaffold to enhance bone defect repair. Osteoprogenitor cells were selected from rat bone marrow stem cells cultured in DMEM medium supplemented with FCS, antibiotics, ascorbic acid, beta-glycerophosphate, and dexamethasone. The selected osteogenic subpopulation was identified by osteocalcin immunohistochemistry as well as Alizarin red S and von Kossa staining which are specific for bone matrix and mineral deposition. Committed osteoprogenitor cells cultured on the hydrogel scaffold were transplanted into the area of a rat tibia segmental bone defect and examined after 6 weeks. Radiology images revealed that 6 weeks post-implantaion, calcified material was present in the site of the defect, indicating new bone formation. It is concluded that committed osteogenic MSCs contained in a biocompatible scaffold can provide a promising surgical tool for enhancement of bone defect healing that will minimize the complications of bone repair in aging and disease. PMID:15621208

Srouji, S; Livne, E

2005-02-01

165

Human Bone Marrow Stromal Cell: Coexpression of Markers Specific for Multiple Mesenchymal Cell Lineages1  

Microsoft Academic Search

The role of hematopoietic stem cells in blood cell development is reasonably understood, whereas the identity and the function of bone marrow stromal cells are much less clear. Using stromal cells in bone marrow cultures of the Dexter type, a favorite medium for the study of hematopoiesis, we show that stromal cells actually represent a unique cell type. Conventional wisdom

Beerelli Seshi; Sanjay Kumar; Debra Sellers

2000-01-01

166

ES cell-derived neuroepithelial cell cultures.  

PubMed

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells. Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5). PMID:18704173

Karki, Shreeya; Pruszak, Jan; Isacson, Ole; Sonntag, Kai C

2006-12-01

167

Optimizing stem cell culture.  

PubMed

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

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

2010-11-01

168

Growth of erythroid burst-forming units (BFU-E) in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs  

SciTech Connect

Erythroid burst-forming units (BFU-E) from canine bone marrow and peripheral blood could be grown in methylcellulose in the presence of an appropriate batch of fetal calf serum (FCS), transferrin, and erythropoietin (Epo). However, improved colony formation (size and number of bursts) was obtained when serum from total body irradiated dogs was present in the culture. This serum, obtained from dogs at day 9 after total body irradiation with a dose of 3.9 Gy, reduced markedly the Epo requirement of BFU-E. Furthermore, it allowed the omission of FCS from the culture medium if cholesterol and bovine serum albumin (BSA) were used as FCS substitutes. BFU-E concentrations were found to be rather different in the peripheral blood and in bone marrow samples from different sites (i.e., iliac crest, sternum, and humerus) of normal beagles. The studies further show that canine bone marrow BFU-E can be cryopreserved in liquid nitrogen.

Kreja, L.; Baltschukat, K.; Nothdurft, W.

1988-08-01

169

Growth of erythroid burst-forming units (BFU-E) in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs.  

PubMed

Erythroid burst-forming units (BFU-E) from canine bone marrow and peripheral blood could be grown in methylcellulose in the presence of an appropriate batch of fetal calf serum (FCS), transferrin, and erythropoietin (Epo). However, improved colony formation (size and number of bursts) was obtained when serum from total body irradiated dogs was present in the culture. This serum, obtained from dogs at day 9 after total body irradiation with a dose of 3.9 Gy, reduced markedly the Epo requirement of BFU-E. Furthermore, it allowed the omission of FCS from the culture medium if cholesterol and bovine serum albumin (BSA) were used as FCS substitutes. BFU-E concentrations were found to be rather different in the peripheral blood and in bone marrow samples from different sites (i.e., iliac crest, sternum, and humerus) of normal beagles. The studies further show that canine bone marrow BFU-E can be cryopreserved in liquid nitrogen. PMID:3292279

Kreja, L; Baltschukat, K; Nothdurft, W

1988-08-01

170

Optimizing stem cell culture  

PubMed Central

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

Van Der Sanden, Boudewijn; Dhobb, Mehdi; Berger, Francois; Wion, Didier

2010-01-01

171

Mechanism of antitumor activity of bone marrow natural suppressor cells.  

PubMed

Co-culturing of P-815 tumor cell strain and intact mouse bone marrow cells nonadherent to plastic resulted in the appearance of soluble mediators with antitumor activity. Bone marrow cells start releasing these antiproliferative factors only after signal exchange with the target tumor cells. The cell-cell contact is an important factor for the induction of antitumor activity. Antitumor activity of bone marrow cells (similarly as immunosuppressive activity) is realized through suppressor factors; the appearance of these factors is induced by target tumor cells. PMID:16027817

Bel'skii, Yu P; Patrushev, V K; Bel'skaya, N V; Danilets, M G; Trofimova, E S; Agafonov, V I

2005-02-01

172

Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material, consisting of poly- l-lactide, carbon nanotubes and microhydroxyapatite, in the presence of bone morphogenetic protein-2  

Microsoft Academic Search

In bone tissue engineering composite materials have been introduced, combining a degradable polymer matrix with, for instance, carbon nanotubes (CNTs) to improve mechanical properties or with microhydroxyapatite (?HA) to improve osteoconduction. The addition of bone morphogenetic protein-2 (BMP-2) can further improve the biological response to the material. However, the influence of such an elaborate composite formation on osteoprogenitor cells is

Meike van der Zande; X. Frank Walboomers; Mathias Brännvall; Beatriz Olalde; Maria J. Jurado; J. Iñaki Álava; John A. Jansen

2010-01-01

173

Chondrogenic differentiation of murine C3H10T1\\/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures  

Microsoft Academic Search

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1\\/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1\\/2 cells have been previously shown to undergo multiple

Andrew E. Denker; Andrew R. Haas; Steven B. Nicoll; Rocky S. Tuan

1999-01-01

174

ACTH promotes chondrogenic nodule formation and induces transient elevations in intracellular calcium in rat bone marrow cell cultures via MC2-R signaling  

PubMed Central

Adrenocorticotropic hormone (ACTH) is among the several melanocortin peptide hormones that are derived from proopiomelanocortin (POMC). ACTH has been found to enhance osteogenesis and chondrogenesis. We show that in the presence of dexamethasone, ACTH dose-dependently increases chondrogenic nodule formation in bone marrow stromal cells (BMSC) from the Wystar Kyoto (WKY) rat. The nodules consist of condensed cells highly expressing alkaline phosphatase, Sox9 and type II collagen transcripts, and a proteoglycan rich matrix. Immunoblot analysis of crude membrane fractions was used to determine that these cells express three melanocortin receptors (MC-R); MC2-R, MC3-R and MC5-R, as well as the melanocortin 2-receptor accessory protein (MRAP). To determine which of these receptors mediate ACTH-induced effects, we used MC-R specific peptides and the known agonist profiles of the receptors. Neither ?-MSH, a strong agonist of the MC5-R nor ?2-MSH, a strong agonist of the MC3-R, duplicates ACTH effects in rat BMSC. In addition, calcium flux was examined as a mechanism for ACTH action at the MC2-R. Consistent with MC2-R and MRAP expression patterns in the BMSC cultures, ACTH-induced transient increases in intracellular calcium were increased with dexamethasone treatment. Neither ?-MSH nor ?2-MSH affected calcium flux. Dexamethasone increased MC2-R and MRAP expression as well as POMC peptide expression and cleavage to increase the production of the lipolytic ?-LPH product. Therefore the effects of ACTH in rat BMSC enriched for mesenchymal progenitors are consistent with an MC2-R signaling mechanism and dexamethasone is capable of regulating components of the melanocortin system in these cells.

Evans, Jodi F.; Rodriguez, Sylvana; Ragolia, Louis

2013-01-01

175

Bone Marrow Stromal Cell-Derived Vascular Endothelial Growth Factor (VEGF) Rather Than Chronic Lymphocytic Leukemia (CLL) Cell-Derived VEGF Is Essential for the Apoptotic Resistance of Cultured CLL Cells  

PubMed Central

Chronic lymphocytic leukemia (CLL) cells feature a pronounced apoptotic resistance. The vascular endothelial growth factor (VEGF) possesses a role in this apoptotic block, although underlying functional mechanisms and the involvement of the microenvironment are unclear. In this study, the VEGF status in CLL was assessed by enzyme-linked immunosorbent assay and immunofluorescence. VEGF receptor 2 (VEGFR2) phosphorylation was determined flow cytometrically and by immunofluorescence. For co-culture, CLL cells were cultivated on a monolayer of the bone marrow–derived stromal cell (BMSC) line HS5. Secreted VEGF was neutralized using the monoclonal antibody mAb293 (R&D Systems, Minneapolis, MN, USA). To block protein secretion, we used Brefeldin A. VEGF was downregulated in BMSCs by small interfering RNA (siRNA), and we assessed survival by annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. CLL cells express and secrete VEGF and possess phosphorylated VEGFR2. This positive VEGF status is not sufficient to prevent spontaneous apoptosis in vitro. Coculture with BMSCs, which secrete vast amounts of VEGF, maintains in vitro CLL cell survival. Blockage of secreted VEGF using the monoclonal antibody mAb293 significantly reduced the survival support for cocultured CLL cells. Both general blockage of protein secretion by Brefeldin A in BMSCs, but not in CLL cells, and siRNA-mediated downregulation of VEGF in BMSCs, significantly reduced the coculture-mediated survival support for CLL cells. It can be concluded that BMSC-derived proteins and VEGF, in particular, but not CLL cell–derived VEGF, is essentially involved in the coculture-mediated survival support for CLL cells. Hence, therapeutic targeting of VEGF signaling might be a promising approach to overcome the apoptotic resistance CLL cells feature within their natural microenvironment.

Gehrke, Iris; Gandhirajan, Rajesh Kumar; Poll-Wolbeck, Simon Jonas; Hallek, Michael; Kreuzer, Karl-Anton

2011-01-01

176

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells  

Microsoft Academic Search

One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion

S N Robinson; J Ng; T Niu; H Yang; J D McMannis; S Karandish; I Kaur; P Fu; M Del Angel; R Messinger; F Flagge; M de Lima; W Decker; D Xing; R Champlin; E J Shpall

2006-01-01

177

Cell isolation and culture.  

PubMed

Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of resolution that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. elegans cells from both embryos and all four larval stages. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior. This chapter describes protocols for the isolation and culture of C. elegans embryonic and larval stage cells. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices. PMID:23430760

Zhang, Sihui; Kuhn, Jeffrey R

2013-01-01

178

Novel three-dimensional long-term bone marrow culture system using polymer particles with grafted epoxy-polymer-chains supports the proliferation and differentiation of hematopoietic stem cells.  

PubMed

Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34(+) cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34(+) cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell-stromal cell interactions. PMID:22016397

Hirabayashi, Yukio; Hatta, Yoshihiro; Takeuchi, Jin; Tsuboi, Isao; Harada, Tomonori; Ono, Kentaro; Glomm, Wilhelm Robert; Yasuda, Masahiro; Aizawa, Shin

2011-11-01

179

Generation of Large Numbers of Dendritic Cells from Mouse Bone Marrow Cultures Supplemented with Granulocyte\\/Macrophage Colony-stimulating Factor  

Microsoft Academic Search

Summary Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow.

Kayo Inaba; Muneo Inaba; Nikolaus Romani; Hideki Aya; Masashi Deguchi; Susumu Ikehara; Shigeru Muramatsu; Ralph M. Steinmanll

1992-01-01

180

Proliferation of human hematopoietic bone marrow cells in simulated microgravity  

Microsoft Academic Search

Summary  Expansion and\\/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle\\u000a in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity\\u000a (?-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the\\u000a field of stem cell biology in

P. Artur Plett; Stacy M. Frankovitz; Rafat Abonour; Christie M. Orschell-Traycoff

2001-01-01

181

Induction of matrix metalloproteinases MMP-1 and MMP-2 by co-culture of breast cancer cells and bone marrow fibroblasts  

Microsoft Academic Search

Two invasive breast cancer cell lines (MDA-MB-231 and BT-549) were found to be more adherent and have greater migratory capacity on bone marrow fibroblasts than three non-invasive cell lines (MCF-7, T47D and BT-483). Antibodies to the adhesion molecules CD44, E-cadherin, ICAM-1, and integrin chains a2, a3, a4, a5, a6, av, a1, a3 and a7 failed to inhibit breast cancer cell

Sonia Saad; Linda J Bendall; Alexander James; David J Gottlieb; Kenneth F Bradstock

2000-01-01

182

Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPAR?2 and increasing Cbfa1\\/Runx2 expression in bone marrow mesenchymal cell cultures  

Microsoft Academic Search

The mechanism whereby lovastatin can counteract steroid-induced osteonecrosis and osteoporosis is poorly understood. We assessed the effect of lovastatin on a multipotential cell line, D1, which is capable of differentiating into either the osteoblast or the adipocyte lineage. The expression of bone cell and fat cell transcription factors Cbfa1\\/Runx2 and PPAR?2, respectively, were determined. 422aP2 gene expression was analyzed. Osteocalcin

Xudong Li; Quanjun Cui; Chinghai Kao; Gwo-Jaw Wang; Gary Balian

2003-01-01

183

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

SciTech Connect

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

Miettinen, Johanna A., E-mail: johanna.miettinen@oulu.fi [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Pietilae, Mika [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland)] [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Salonen, Riikka J. [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland) [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Ohlmeier, Steffen [Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 Oulu (Finland)] [Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 Oulu (Finland); Ylitalo, Kari; Huikuri, Heikki V. [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland)] [Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland)] [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu (Finland)

2011-04-01

184

Evaluation Of Myelotoxicity In Cotton Rats (Sigmodon hispidus) Exposed To Environmental Contaminants. I. In Vitro Bone-Marrow Progenitor Culture  

Microsoft Academic Search

Bone marrow is extremely sensitive to toxicants, and in vitro culture of bone-marrow progenitor cells has been shown to be a sensitive indicator of bone-marrow injury in laboratory rodents. The ability of a bone-marrow progenitor cell assay to detect myelotoxicity in a wild rodent model (cotton rat, Sigmodon hispidus) that inhabits many contaminated ecosystems in the southern United States was

Soochong Kim; Eric L. Stair; Robert L. Lochmiller; James W. Lish; Charles W. Qualls Jr

2001-01-01

185

Mammalian Cell Culture Simplified.  

ERIC Educational Resources Information Center

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

Moss, Robert; Solomon, Sondra

1991-01-01

186

Enhanced proliferation of bone marrow mesenchymal stem cells by co-culture with TM4 mouse Sertoli cells: involvement of the EGF/PI3K/AKT pathway.  

PubMed

Bone marrow mesenchymal stem cells (BM-MSCs) are considered as a promising option in the field of regenerative medicine and tissue engineering. However, little is known about how TM4 mouse Sertoli cells, which are known to enhance stem cells proliferation in co-culture, may influence the proliferation of BM-MSCs and which signaling pathways are involved in. To address these questions, an in vitro transwell system was used. We found that TM4 cells could produce soluble factors which enhanced the growth of BM-MSCs without inhibiting the multipotency. Furthermore, cell cycle analysis showed that co-culture with the TM4 cells accelerated the progress of BM-MSCs from the G1 to the S phase. The expression of the phospho-akt, mdm2, as well as pho-CDC2, and cyclin D1 were markedly upregulated in co-cultured BM-MSCs. The observed promoting effect was significantly inhibited by the administration of the PI3K/AKT inhibitor, LY294002. Among the various growth factors produced by TM4 cells, the epithelial growth factor (EGF) stimulated the proliferation of the BM-MSCs more significantly compared with the other growth factors examined in this study. Neutralization of EGF via a blocking antibody significantly limited the promoting growth effect in BM-MSCs. These results suggest that TM4 cells provide a favorable in vitro environment for BM-MSCs growth and imply the involvement of the EGF/PI3K/AKT pathway. PMID:24748323

Tian, Huan; Guo, Meijin; Zhuang, Yingping; Chu, Ju; Zhang, Siliang

2014-08-01

187

Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacity.  

PubMed

The ability of bone marrow stromal cells (BMSCs) to differentiate into osteoblasts is being exploited in cell-based therapy for repair of bone defects. However, the phenotype of ex vivo cultured BMSCs predicting their bone-forming capacity is not known. Thus we employed DNA microarrays comparing two human bone marrow stromal cell (hBMSC) populations: One is capable of in vivo heterotopic bone formation (hBMSC-TERT(+Bone)), and the other is not (hBMSC-TERT(-Bone)). Compared with hBMSC-TERT(-Bone), the hBMSC-TERT(+Bone) cells had an increased overrepresentation of extracellular matrix genes (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor-binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT(-Bone) cells expressed a larger number of immune-response-related genes (26% versus 8%). In order to test for the predictive value of these markers, we studied the correlation between their expression levels in six different hBMSC-derived clones and the ability to form bone in vivo. We found a significant correlation for decorin, lysyl oxidase-like 4, natriuretic peptide receptor C, and tetranectin. No significant positive correlation was found for canonical osteoblastic markers Runx2, alkaline phosphatase, collagen type I, osteopontin, and bone sialoprotein. Prospective isolation of four additional hBMSC clones based on their expression levels of the molecular markers correlated with their in vivo bone-formation ability. In conclusion, our data suggest an in vitro molecular signature predictive for hBMSCs' in vivo bone-formation ability. Identifying more of these predictive markers would be very useful in the quality control of osteoblastic cells before use in therapy. PMID:19821776

Larsen, Kenneth H; Frederiksen, Casper M; Burns, Jorge S; Abdallah, Basem M; Kassem, Moustapha

2010-04-01

188

[Pluripotency of bone marrow stromal cells and perspectives of their use in cell therapy].  

PubMed

The possibility of differentiation of insulin-producing cells and neural and glial elements was demonstrated in the culture of bone marrow stromal cells. The perspectives of use of the bone marrow stromal cells in clinical medicine are considered. PMID:12816054

Shchegel'skaia, E A; Mikulinski?, Iu E; Revishchin, A V; Omel'chenko, E A; Kul'shin, V E; Grishchenko, V I; Korochkin, L I

2003-01-01

189

[Bone and Stem Cells. Mesenchymal stem cells and bone regeneration].  

PubMed

Mesenchymal stem cells (MSCs) have multi-differentiation potency, and enhance wound healing in various kinds of disease. Recently MSC not only differentiate into tissue-forming cells, but also secrete various kinds of cytokines and chemokines that are anti-apoptotic, immunomodulatory, angiogenic, and the cell-mobilizing to influence extracellular environment. In addition, we show that MSC has a novel intercellular communication mechanism. It hopes to suggest ways to make safer and reliable usage of MSC in bone regeneration. PMID:24681503

Komaki, Motohiro; Iwasaki, Kengo; Morita, Ikuo

2014-04-01

190

Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate  

SciTech Connect

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.

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

1989-11-25

191

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

PubMed Central

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.

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

192

Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system  

Microsoft Academic Search

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrix®

Yi-Chin Toh; Saey Tuan Ho; Yi Zhou; Dietmar W. Hutmacher; Hanry Yu

2005-01-01

193

Mineralization and bone formation on microcarrier beads with isolated rat calvaria cell population  

Microsoft Academic Search

Using enzymatically isolated rat bone cells in the presence of cytodex microcarrier beads, osteoblastic cell differentiation and bone nodule formation were studied at the optical and electron microscopic level. Cytochemical method showed an intense alkaline phosphatase activity mainly around the microcarriers where the cells have formed multilayers on day 4 of cultures. On day 7 of experiment cultures. Von Kossa

Jean-Michel Sautier; Jean-Raphaël Nefussi; Nadine Forest

1992-01-01

194

Osteoclast development: the cell surface and the bone environment.  

PubMed

Bone development and remodelling processes depend on complex interactions between bone cell precursors, mature bone cells, extracellular matrix molecules, growth factors, the immune system and humoral factors. The exact molecular nature of many of the cell-cell and cell-matrix interactions occurring during bone remodelling remains to be resolved. Cell surface molecules are likely to have important roles in both bone cell differentiation and regulatory processes. However, little is known about changes in the osteoclast cell surface during development and there is only limited information on the cell surface composition of the mature cell phenotype. We describe how one osteoclast-specific monoclonal antibody has been used to identify, characterize and purify a 96 kDa/140 kDa osteoclast membrane protein. The antibody has also been used as a phenotypic marker in studies designed to identify soluble and matrix-related bone factors involved in the terminal stages of osteoclast differentiation. In parallel studies using marrow-derived giant cells and the chick chorioallantoic membrane (CAM), immunohistochemical and enzyme-linked immunoassays (ELISA) have been used to investigate the influence of calvaria, calvaria-conditioned medium, bone matrix, and bone matrix components on osteoclast development. Marrow-derived giant cells express osteoclast-specific cell surface antigens when co-cultured with live calvariae or when exposed to calvaria-conditioned medium. In the richly vascularized and mesenchymal cell-containing CAM, intact bone matrix induces the formation of giant cells that express the osteoclast-specific antigens. In contrast, isolated bone matrix components implanted on the CAM recruit only mononuclear cells which are not recognized by the osteoclast-specific antibody. PMID:3068005

Osdoby, P; Oursler, M J; Salino-Hugg, T; Krukowski, M

1988-01-01

195

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

PubMed

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

Zubov, D O

2008-01-01

196

Culture conditions allow selection of different mesenchymal progenitors from adult mouse bone marrow.  

PubMed

The use of adult stem cells in tissue engineering approaches will benefit from the establishment of culture conditions that allow the expansion and maintenance of cells with stem cell-like activity and high differentiation potential. In the field of adult stem cells, bone marrow stromal cells (BMSCs) are promising candidates. In the present study, we define, for the first time, conditions for optimizing the yields of cultures enriched for specific progenitors of bone marrow. Using four distinct culture conditions, supernatants from culture of bone fragments, marrow stroma cell line MS-5, embryonic fibroblast cell line NIH3T3, and a cocktail of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), we isolated four different sub-populations of murine BMSCs (mBMSCs). These cells express a well-known marker of undifferentiated embryonic stem cells (Nanog) and show interesting features in immunophenotype, self-renewal ability, and differentiation potency. In particular, using NIH3T3 conditioned medium, we obtained cells that showed impairment in osteogenic and chondrogenic differentiation while retaining high adipogenic potential during passages. Our results indicate that the choice of the medium used for isolation and expansion of mBMSCs is important for enriching the culture of desired specific progenitors. PMID:19298168

Esposito, Maria Teresa; Di Noto, Rosa; Mirabelli, Peppino; Gorrese, Marisa; Parisi, Silvia; Montanaro, Donatella; Del Vecchio, Luigi; Pastore, Lucio

2009-09-01

197

Molluscan cells in culture: primary cell cultures and cell lines  

PubMed Central

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

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

2013-01-01

198

Repaire of Large Segmental Bone Defects by Marrow Stromal Cells Transplant with Demineralized Bone Matrix  

Microsoft Academic Search

The aim of this study was to determine the healing potential of large segmental bone defects using tissue-engineered constructs in a large animal model before it can be introduced into a clinical trial. Isolated Autologous marrow stromal cells (aMSCs) were cultured in vitro, then seeded into allogenic demineralized bone matrix (aDBM) and co-cultivated for 7 days to construct DBM-MSCs complex.

Zhao Xie; Fei Luo; Jian-zhong Xu; Shi-wu Dong

2008-01-01

199

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

PubMed

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

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

2014-04-01

200

[Bone and Stem Cells. Intravital imaging of bone marrow microenvironment].  

PubMed

Various kinds of cell types, such as osteoclasts, osteoblasts, hematopoietic cells, and mesenchymal cells, have been reported to exist in the bone marrow and communicate with each other. Although there have been many previous studies about bone marrow microenvironment, most of them were analyzed by conventional methods such as histological analysis and flow cytometry. These methods could not observe the dynamic cell movement in living bone marrow. Recently rapid development of fluorescent imaging techniques enables us to understand the cellular dynamics in vivo . That's why we have originally established an advanced imaging system for visualizing living bone tissues with intravital two-photon microscopy. Here we show the latest data and the detailed methodology of intravital imaging of bone marrow microenvironment, and also discuss its further application. PMID:24681500

Mizuno, Hiroki; Kikuta, Junichi; Ishii, Masaru

2014-04-01

201

Massive bone reconstruction with heat-treated bone graft loaded autologous bone marrow-derived stromal cells and ?-tricalcium phosphate composites in canine models.  

PubMed

Bone marrow-derived stromal cells (BMSCs) contain mesenchymal stem cells that are capable of forming various mesenchymal tissues. We hypothesized that BMSCs and ?-tricalcium phosphate (?-TCP) composites would promote the remodeling of large-sized autologous devitalized bone grafts; therefore, the aim of this study was to evaluate the effects of the composites on the remodeling of autologous devitalized bone grafts. Autologous BMSCs cultured in culture medium containing dexamethasone (10(-7) ?M) were loaded into porous ?-TCP granules under low-pressure. Theses BMSC/TCP composites were put into the bone marrow cavity of autologous heat-treated bone (femoral diaphysis, 65-mm long, 100°C, 30?min) and put back to the harvest site. In the contralateral side, ?-TCP without BMSC were used in the same manner as the opposite side as the control. Treatment with the BMSC/TCP composites resulted in a significant increase in thickness, bone mineral density, and matured bone volume of the cortical bone at the center of the graft compared to the control. Histological analysis showed matured regenerated bone in the BMSC loaded group. These results indicate that BMSC/TCP composites facilitated bone regeneration and maturation at the graft site of large-sized devitalized bone. This method could potentially be applied for clinical use in the reconstruction of large bone defects such as those associated with bone tumors. PMID:23589164

Koyanagi, Hirotaka; Ae, Keisuke; Maehara, Hidetsugu; Yuasa, Masato; Masaoka, Tomokazu; Yamada, Tsuyoshi; Taniyama, Takashi; Saito, Masanori; Funauchi, Yuki; Yoshii, Toshitaka; Okawa, Atsushi; Sotome, Shinichi

2013-08-01

202

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

PubMed Central

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

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

2012-01-01

203

Microarray Analysis of Human Adipose-Derived Stem Cells in Three-Dimensional Collagen Culture: Osteogenesis Inhibits Bone Morphogenic Protein and Wnt Signaling Pathways, and Cyclic Tensile Strain Causes Upregulation of Proinflammatory Cytokine Regulators and Angiogenic Factors  

PubMed Central

Human adipose-derived stem cells (hASC) have shown great potential for bone tissue engineering. However, the molecular mechanisms underlying this potential are not yet known, in particular the separate and combined effects of three-dimensional (3D) culture and mechanical loading on hASC osteogenesis. Mechanical stimuli play a pivotal role in bone formation, remodeling, and fracture repair. To further understand hASC osteogenic differentiation and response to mechanical stimuli, gene expression profiles of proliferating or osteogenically induced hASC in 3D collagen I culture in the presence and absence of 10% uniaxial cyclic tensile strain were examined using microarray analysis. About 847 genes and 95 canonical pathways were affected during osteogenesis of hASC in 3D culture. Pathway analysis indicated the potential roles of Wnt/?-catenin signaling, bone morphogenic protein (BMP) signaling, platelet-derived growth factor (PDGF) signaling, and insulin-like growth factor 1 (IGF-1) signaling in hASC during osteogenic differentiation. Application of 10% uniaxial cyclic tensile strain suggested synergistic effects of strain with osteogenic differentiation media on hASC osteogenesis as indicated by significantly increased calcium accretion of hASC. There was no significant further alteration in the four major pathways (Wnt/?-catenin, BMP, PDGF, and IGF-1). However, 184 transcripts were affected by 10% cyclic tensile strain. Function and network analysis of these transcripts suggested that 10% cyclic tensile strain may play a role during hASC osteogenic differentiation by upregulating two crucial factors in bone regeneration: (1) proinflammatory cytokine regulators interleukin 1 receptor antagonist and suppressor of cytokine signaling 3; (2) known angiogenic inductors fibroblast growth factor 2, matrix metalloproteinase 2, and vascular endothelial growth factor A. This is the first study to investigate the effects of both 3D culture and mechanical load on hASC osteogenic differentiation. A complete microarray analysis investigating both the separate effect of soluble osteogenic inductive factors and the combined effects of chemical and mechanical stimulation was performed on hASC undergoing osteogenic differentiation. We have identified specific genes and pathways associated with mechanical response and osteogenic potential of hASC, thus providing significant information toward improved understanding of our use of hASC for functional bone tissue engineering applications.

Charoenpanich, Adisri; Wall, Michelle E.; Tucker, Charles J.; Andrews, Danica M.K.; Lalush, David S.

2011-01-01

204

Osteopenic bone cell response to strontium-substituted hydroxyapatite  

Microsoft Academic Search

Ionic substitution is a powerful tool to improve the biological performance of calcium phosphate based materials. In this\\u000a work, we investigated the response of primary cultures of rat osteoblasts derived from osteopenic (O-OB) bone to strontium\\u000a substituted hydroxyapatite (SrHA), and to hydroxyapatite (HA) as reference material, compared to normal (N-OB) bone cells.\\u000a Strontium (Sr) and calcium (Ca) cumulative releases in

E. Boanini; P. Torricelli; M. Fini; A. Bigi

205

Murine bone marrow cell line producing colony-stimulating factor  

SciTech Connect

A cell line (H-1) derived from the adherent layer of a 14-wk-old Dexter bone marrow culture has been maintained as cloned and uncloned lines through 21 passages at the time of these studies. These cell lines develop many fat droplets as they age and become confluent. The uncloned line produces increasing amounts of colony-stimulating activity as the cells become confluent. Feeder-layers or supernatants from the nonconfluent or confluent fat-laden cells stimulate the formation of greater numbers of colonies derived from cultures of colony-forming units (CFU) than does medium from L cell culture containing colony-stimulating factor (CSF). Antibody to the CSF-containing medium from L cell culture neutralizes the colony-stimulating activity, thus showing immunologic similarity to a known molecular species that stimulates colony production in a CFU culture that produces granulocyte or macrophage populations, or both.

Harigaya, K. (Brookhaven National Lab., Upton, NY); Cronkite, E.P.; Miller, M.E.; Shadduck, R.K.

1981-11-01

206

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

PubMed Central

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 45Ca release from 45Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with 45Ca-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, from 27 +/- 2% (mean +/- SEM) 45Ca release in cultures with no added cadmium to 68 +/- 6% release in cultures containing cadmium (n = 4). 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. Images

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

1988-01-01

207

Bone cell kinetics during longitudinal bone growth in the rat  

Microsoft Academic Search

Summary  The purpose of this work was to provide further knowledge about bone cell kinetics in the metaphysis of the growing long bone.\\u000a Seventy rats were sacrificed from 1 to 120 h after injection of tritiated thymidine. Autoradiographs of 3 µm thick sections\\u000a of the proximal tibial metaphysis were studied in a manner which allowed evaluation of labeled cell nuclei as

Donald B. Kimmel; Webster S. S. Jee

1980-01-01

208

Bone formation in rabbit cancellous bone explant culture model is enhanced by mechanical load  

PubMed Central

Background When studying and designing an artificial bone in vitro with similar features and functionality of natural bone by tissue engineering technology, the culturing environment, especially the mechanical environment is supposed to be an important factor, because a suitable mechanical environment in vitro may improve the adaptability of the planted-in tissue engineering bone in the body. Unfortunately, up to now, the relationship between mechanical stimuli and natural bone growth has not yet been precisely determined, and it is so imperative for a prior study on effect of mechanical loading on growth of the natural bone cultured in vitro. Methods Under sterile conditions, explant models of rabbit cancellous bone with 3?mm in thickness and 8?mm in diameter were prepared and cultured in a dynamic loading and circulating perfusion bioreactor system. By Micro-CT scanning, a 3D model for finite element (FEM) analysis was achieved. According to the results of FEM analysis and physiological load bearing capacity of the natural bone, these models were firstly subjected to mechanical load with 1Hz frequency causing average apparent strain of 1000 ??, 2000 ??, 3000 ?? and 4000 ?? respectively for 30?min every day, activities of alkaline phosphatase (AKP) were detected on the 5th and the 14th loading day and on the 14th and the 21st day, mechanical properties, tissue mineral density (TMD) of the bone explant models were investigated and Von-kossa staining and fluorescence double labeling assays were conducted to evaluate whether there were fresh osteoid in the bone explant models. In addition, Western blot, Elisa and Real-time PCR were employed to analyze expression of Collagen-I (COL-1), bone morphogenetic protein-2 (BMP-2) and osteoprotegerin (OPG) protein and RNA. Results The explant models of rabbit cancellous bone prepared under sterile conditions grew well in the bioreactor system. With the increasing culturing time and load levels, bone explant models in groups with 1000 ?? and 2000 ?? average apparent strain experienced improving mechanical properties and TMD (P<0.05), and results of Von-kossa staining and fluorescence double labeling also showed apparent fresh osteoid formation. Under the same loading conditions, a up-regulations in protein and RNA of COL-1, BMP-2 and OPG were detected, especially, relative genes notably expressed after 21?days. Conclusion Our study demonstrated that mechanical load could improve function and activity of osteoblasts in explant models of cancellous bone. Through regulations of COL-1, OPG and BMP-2 secreted by osteoblasts, the mechanical load could improve the tissue structural density and stiffness due to formation of fresh osteoid.

2013-01-01

209

Homing of Cancer Cells to the Bone  

Microsoft Academic Search

A variety of tumor cells preferentially home to the bone. The homing of cancer cells to the bone represents a multi-step process\\u000a that involves malignant progression of the tumor, invasion of the tumor through the extracellular matrix and the blood vessels\\u000a and settling of the tumor cells in the bone. Gaining a greater understanding as to the mechanisms used by

Anjali Mishra; Yusuke Shiozawa; Kenneth J. Pienta; Russell S. Taichman

210

In vivo bone metastases, osteoclastogenic ability, and phenotypic characterization of human breast cancer cells  

Microsoft Academic Search

Mouse bone marrow cells cultured with human breast cancer MCF-7 cell-conditioned media showed osteoclastogenesis with an increment of bone resorption, although conditioned media from an adriamycin-selected MCF-7 clone (MCF-7ADR) had no effect. Consistently, MCF-7 cells induced 5-fold more in vivo experimental osteolytic bone metastases, with no soft tissue lesions, compared to MCF-7ADR cells. Paracrine factors stimulating (interleukin (IL)-6, IL-1?, tumor necrosis

Nadia Rucci; Enrico Ricevuto; Corrado Ficorella; Maurizio Longo; Marie Perez; Claudia Di Giacinto; Alessia Funari; Anna Teti; Silvia Migliaccio

2004-01-01

211

Homing of cancer cells to the bone.  

PubMed

A variety of tumor cells preferentially home to the bone. The homing of cancer cells to the bone represents a multi-step process that involves malignant progression of the tumor, invasion of the tumor through the extracellular matrix and the blood vessels and settling of the tumor cells in the bone. Gaining a greater understanding as to the mechanisms used by cancer cells in these processes will facilitate the design of drugs which could specifically target the homing process. In this review we will discuss the properties of tumor cells and the bone microenvironment which promote homing of a cancer cell to the bone. We will highlight the different steps and the molecular pathways involved when a cancer cell metastasize to the bone. Since bone is the major home for hematopoietic stem cells (HSCs), we will also highlight the similarities between the homing of cancer and HSC to the bone. Finally we will conclude with therapeutic and early detection strategies which can prevent homing of a cancer cell to the bone. PMID:21826451

Mishra, Anjali; Shiozawa, Yusuke; Pienta, Kenneth J; Taichman, Russell S

2011-12-01

212

Cell therapy in bone healing disorders  

PubMed Central

In addition to osteosynthetic stabilizing techniques and autologous bone transplantations, so-called orthobiologics play an increasing role in the treatment of bone healing disorders. Besides the use of various growth factors, more and more new data suggest that cell-based therapies promote local bone regeneration. For ethical and biological reasons, clinical application of progenitor cells on the musculoskeletal system is limited to autologous, postpartum stem cells. Intraoperative one-step treatment with autologous progenitor cells, in particular, delivered promising results in preliminary clinical studies. This article provides an overview of the rationale for, and characteristics of the clinical application of cell-based therapy to treat osseous defects based on a review of existing literature and our own experience with more than 100 patients. Most clinical trials report successful bone regeneration after the application of mixed cell populations from bone marrow. The autologous application of human bone marrow cells which are not expanded ex vivo has medico-legal advantages. However, there is a lack of prospective randomized studies including controls for cell therapy for bone defects. Autologous bone marrow cell therapy seems to be a promising treatment option which may reduce the amount of bone grafting in future.

Jager, Marcus; Hernigou, Philippe; Zilkens, Christoph; Herten, Monika; Li, Xinning; Fischer, Johannes; Krauspe, Rudiger

2010-01-01

213

1009. High Expression of Foxp3 mRNA in Rat Dendritic Cells (DCs) Generated from a Long-Term Culture of Bone Marrow Cells (BMC)  

Microsoft Academic Search

Foxp3 is recognized as to encode a transcription suppressor with a forkhead\\/winged helix motif at the carboxy terminus, a C2H2 zinc finger domain, and a 3-heptad Zip motif.Heretofore, numerous studies have been reported that Scurfin , the protein product of the FoxP3 gene, is a marker of regulatory T cells that negatively regulates T cell function.Thus it has been reported

Atsushi Tsuji; Li Xaio-Kang; Hiromitsu Kimura

2006-01-01

214

Humanized culture of periosteal progenitors in allogeneic serum enhances osteogenic differentiation and in vivo bone formation.  

PubMed

The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration. PMID:24375540

Roberts, Scott J; Owen, Helen C; Tam, Wai Long; Solie, Lien; Van Cromphaut, Sophie J; Van den Berghe, Greet; Luyten, Frank P

2014-02-01

215

Cell culture's spider silk road.  

PubMed

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

Perkel, Jeffrey

2014-01-01

216

Catecholamines in murine bone marrow derived mast cells  

Microsoft Academic Search

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753±844 pg\\/107 cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, ?-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine ?10 ?M affected IL-3-induced cell proliferation. High exogenous dopamine (20–100 ?M) decreased

Jessica G Freeman; John J Ryan; Christopher P Shelburne; Daniel P Bailey; L. Andrew Bouton; Nedathur Narasimhachari; Jos Domen; Nathalie Siméon; François Couderc; Jennifer K Stewart

2001-01-01

217

Physiological effects of microgravity on bone cells.  

PubMed

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

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

2014-06-01

218

Specific effect of immunomodulatory quinoline-3-carboxamide ABR-215757 in GM-CSF stimulated bone marrow cell cultures: Block of initiation of proliferation of Gr1 + cells  

Microsoft Academic Search

Quinoline-3-carboxamides are currently in clinical development for treatment of both autoimmune disease and cancer. Carboxamides such as ABR-215757 (5757) have shown efficacy in several in vivo mouse models of human inflammatory autoimmune disease. Some microbial infections in mice cause GM-CSF dependent accumulation of dendritic cells expressing TNF? and inducible nitric oxide synthase (iNOS; Tip-DCs) in lymphoid organs. Functionally similar DCs

Sofia Helmersson; Martin Stenström; Tomas Leanderson; Fredrik Ivars

2011-01-01

219

Interactions of total bone marrow cells with increasing quantities of macroporous calcium phosphate ceramic granules.  

PubMed

The biological properties of synthetic calcium phosphate bioceramics have made them the third choice of material for bone reconstructive surgery, after autologous bone and allografts. Nevertheless, bioceramics lack the osteogenic properties that would allow them to repair large bone defects. One strategy in bone tissue engineering consists of associating a synthetic scaffold with osteogenic cells. Mesenchymal stem cells (MSC) are usually isolated from bone marrow cultured for several weeks and seeded on to a small quantity of bioceramic. We have studied the association of total bone marrow cells, harvested from femurs of rats, with increasing amounts of calcium phosphate ceramic granules (50-250 mg). A cell viability test indicated that a little quantity of bioceramics granules (50 mg) was less detrimental for culturing 1 million nucleated cells from the whole bone marrow population. Cell morphology, viability, adhesion and differentiation were studied after different culture periods. Among the heterogeneous population of bone marrow cells, only a limited amount of cells attached and differentiated on the bioceramics. To explain the influence of the amount of synthetic scaffold on cell viability, media calcium concentrations were measured. Low cell viability could be explained by calcium phosphate precipitation leading to a decrease in calcium concentrations observed with relatively large amounts of scaffold. This study showed that the chemical stability of the ceramic plays a critical role in the viability of bone marrow cells. PMID:17554601

Le Nihouannen, Damien; Duval, Laure; Lecomte, Antoine; Julien, Marion; Guicheux, Jérôme; Daculsi, Guy; Layrolle, Pierre

2007-10-01

220

Statistics of hits to bone cell nuclei.  

PubMed

The statistics of hits to the nuclei of bone cells irradiated from alpha sources labeling bone tissue is described. It is shown that the law of remodeling of a bone structural unit (BSU), which describes the distribution of quiescence periods of this unit, affects the statistics of hits. It the irradiation of bone cells occurs during the whole cell cycle, the mean number of hits is independent of the law of remodeling. In this case the variance of hits has the minimum value for constant quiescence periods of BSUs (deterministic remodeling) and the maximum value for exponentially distributed quiescence periods (random remodeling). For the first generation of bone cells, i.e. for the cells which existed at the moment of the uptake of the nuclide, the mean number of hits depends on the law of remodeling. For random remodeling the mean number is equal to the mean value for the complete remodeling cycle. For deterministic remodeling the mean is only half this value. For the first generation of bone cells, changing the law of remodeling from random to deterministic increases the probability of no hits to the nuclei of bone cells. For the same mean value of hits, the difference does not exceed 13.3% of the total number of cells. For the subsequent generations of bone cells, such a change of the law of remodeling decreases the probability of no hits by 20.4%. PMID:8337361

Kruglikov, I L; Polig, E; Jee, W S

1993-01-01

221

[Osteogenic differentiation of human multipotent mesenchymal stromal cells of bone marrow and adipose tissue].  

PubMed

Cellular populations with phenotype similar to multipotent mesenchymal stromal cells have been isolated from two different sources: a human bone marrow and adipose tissue. The comparative analysis of differentiation efficiency of these cells in the direction of osteogenesis revealed morphological changes confirmed by Alizarin red and von Kossa staining in bone marrow cells on the 14th day, and in adipose tissue cells on the 28th day of culturing in the medium with inductors. The analysis of osteopontin, osteocalcin and bone sialoprotein gene expression in RT-PCR reactions detected essential distinctions in the potency of these cells to differentiate into bone tissue cells. PMID:19062517

Savchenkova, I P; Rostovskaia, M S; Chupikova, N I; Sharifullina, S Z; Tepliashin, A S

2008-01-01

222

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

SciTech Connect

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

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

1988-11-01

223

Attachment Formation Following Replantation of Cultured Cells into Periodontal Defects—a Study in Minipigs  

Microsoft Academic Search

Regeneration processes in the periodontium occur by the interaction of different cell populations. It is known that these cells are also capable of forming new periodontal tissue after culture in vitro. The present study investigated whether replanted cultured cells from the periodontium could contribute to attachment formation. Primary cell cultures from alveolar bone and periodontal ligament were obtained from 11

H. Lang; N. Schiuler; R. Nolden

1998-01-01

224

Cell culture purity issues and DFAT cells  

SciTech Connect

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

Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China) [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States)] [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States)] [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China)] [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

2013-04-12

225

Fermentation with immobilized cell cultures.  

PubMed

For the production of monoclonal antibodies and complex recombinant human proteins or glycoproteins a number of immobilized cell culture systems have been developed. The advantages of such cell culture systems are that cells can be kept in small volumes of cell culture fluid and media can be changed continuously if necessary for induction of product synthesis or removal and harvest of metabolic products. Whereas the hollow fiber and the opticell culture systems can be limited in scaling up the microcarrier system, the fluidized bed bioreactor and the solid bed bioreactor are suitable for scaling up. In contrast to the other systems, the solid bed bioreactor requires no special manipulation for anchoring the cells to the wire springs. In situ cleaning is possible and the beads are reusable. With this cell culture fermentation system, production processes for interferon beta, monoclonal antibodies for interferon alfa and recombinant human tissue plasminogen activator were developed. PMID:3285839

Werner, R G; Merk, W; Walz, F

1988-02-01

226

Human preleukaemia cell culture studies in sideroblastic anaemia.  

PubMed Central

Cell structure abnormalties are found in acute leukaemia and preleukaemic states. Studies on bone marrow cells and peripheral leucocytes of 4 patients with idiopathic acquired sideroblastic anaemia showed patterns in cell culture similar to those reported in acute leukaemia: 2 of these patients later developed leukaemia. Other patients with idiopathic, secondary or congenital sideroblastosis showed no such cell culture abnormalities, and none developed leukaemia. Studies such as this suggest that cell culture methods detect altered cellular function preceding overt leukaemia and that these abnormal findings may be helpful in the evaluation of patient groups with an increased incidence of leukaemia.

Senn, J. S.; Pinkerton, P. H.; Price, G. B.; Mak, T. W.; McCulloch, E. A.

1976-01-01

227

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass  

PubMed Central

Aging reduces the number of mesenchymal stem cells (MSCs) in the bone marrow which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct the MSCs to the bone surface by attaching a synthetic high affinity and specific peptidomimetic ligand (LLP2A) against integrin ?4?1 on the MSC surface, to a bisphosphonate (alendronate, Ale) that has high affinity for bone. LLP2A-Ale increased MSCs migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation and immune competent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or following estrogen deficiency. These results provide a proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.

Guan, Min; Yao, Wei; Liu, Ruiwu; Lam, Kit S.; Nolta, Jan; Jia, Junjing; Panganiban, Brian; Meng, Liping; Zhou, Ping; Shahnazari, Mohammad; Ritchie, Robert O.; Lane, Nancy E.

2013-01-01

228

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

PubMed

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

de Peppo, Giuseppe Maria; Marolt, Darja

2014-01-01

229

Ureaplasma infection of cell cultures.  

PubMed Central

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

Kotani, H; McGarrity, G J

1986-01-01

230

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

PubMed Central

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

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

2013-01-01

231

Migration of Co-cultured Endothelial Cells and Osteoblasts in Composite Hydroxyapatite\\/Polylactic Acid Scaffolds  

Microsoft Academic Search

Regeneration of bone in large segmental bone defects requires regeneration of both cortical bone and trabecular bone. A scaffold\\u000a design consisting of a hydroxyapatite (HA) ring surrounding a polylactic acid (PLA) core simulates the structure of bone and\\u000a provides an environment for indirect and direct co-culture conditions. In this experiment, human umbilical vein endothelial\\u000a cells (EC) and normal human primary

Amita R. Shah; Sarita R. Shah; Sunho Oh; Joo L. Ong; Joseph C. Wenke; C. Mauli Agrawal

232

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

PubMed

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

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

2013-03-01

233

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

PubMed Central

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.

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

2013-01-01

234

Basic fibroblast growth factor supports expansion of mouse compact bone-derived mesenchymal stem cells (MSCs) and regeneration of bone from MSC in vivo.  

PubMed

Some progress has been made in development of methods to regenerate bone from cultured cells, however no method is put to practical use. Here, we developed methods to isolate, purify, and expand mesenchymal stem cells (MSCs) from mouse compact bone that may be used to regenerate bone in vivo. These cells were maintained in long-term culture and were capable of differentiating along multiple lineages, including chondrocyte, osteocyte, and adipocyte trajectories. We used standard cell isolation and culture methods to establish cell cultures from mouse compact bone and bone marrow. Cultures were grown in four distinct media to determine the optimal composition of culture medium for bone-derived MSCs. Putative MSCs were subjected to flow cytometry, alkaline phosphatase assays, immunohistochemical staining, and several differentiation assays to assess cell identity, protein expression, and developmental potential. Finally, we used an in vivo bone formation assay to determine whether putative MSCs were capable of regenerating bone. We found that compact bone of mice was a better source of MCSs than the bone marrow, that growth in plastic flasks served to purify MSCs from hematopoietic cells, and that MSCs grown in basic fibroblast growth factor (bFGF)-conditioned medium were, based on multiple criteria, superior to those grown in leukemia inhibitory factor-conditioned medium. Moreover, we found that the MSCs isolated from compact bone and grown in bFGF-conditioned medium were capable of supporting bone formation in vivo. The methods and results described here have implications for understanding MSC biology and for clinical purpose. PMID:22203245

Yamachika, Eiki; Tsujigiwa, Hidetsugu; Matsubara, Masakazu; Hirata, Yasuhisa; Kita, Kenichiro; Takabatake, Kiyofumi; Mizukawa, Nobuyoshi; Kaneda, Yoshihiro; Nagatsuka, Hitoshi; Iida, Seiji

2012-04-01

235

Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture  

Microsoft Academic Search

Aim:To test the hypothesis that genistein stimulates the osteoblastic differentiation through the p38 mitogen activated protein kinase (MAPK)-core-binding factor 1 (Cbfa1) pathway.Methods:The activation of p38 MAPK was detected by Western blotting. Alkaline phosphatase (ALP) activity and calcium deposition were assessed for osteoblastic differentiation of bone marrow-derived mesenchymal stem cell (BMSC) cultures. The expression of Cbfa1 was analyzed at both the

Qing-chuan Liao; Zhou-sheng Xiao; Yan-fang Qin; Hong-hao Zhou

2007-01-01

236

[Bone and Stem Cells. Cellular network in bone micro-environment - histological and ultrastructural aspects -].  

PubMed

Bone micro-environment appears to reflect bone turnover, i.e., frequency of bone remodeling. There are many bone-synthesizing mature osteoblasts, bone-resorbing osteoclasts, and a thick cell layer of preosteoblasts overlying mature osteoblasts in the region which shows active bone remodeling. Bone lining cells, - flattened, resting form of osteoblasts cover the quiescent bone surface, in which, however, osteocyte-lacunar canalicular system tend to be geometrically well-arranged. Thus, bone micro-environment seems to be regulated by preosteoblasts, bone marrow stromal cells and vascular endothelial cells, as well as osteoblasts and osteoclasts. But, precious biological function of preosteoblasts and bone marrow stromal cells are still under the investigation, e.g., due to many phenotypes of preosteoblasts. In this review, we will introduce histological and ultrastructural aspects on cellular involvement in bone micro-environment. PMID:24681493

Amizuka, Norio; Yamamoto, Tomomaya; Hasegawa, Tomoka

2014-04-01

237

Regulation of Tenascin-C Expression in Bone Cells by Transforming Growth Factor-?  

Microsoft Academic Search

Transforming growth factor-? (TGF-?) stimulates new bone formation when administered locally in vivo. The extracellular matrix protein tenascin-C, which is secreted by osteoblasts but absent from mineralized bone matrix, supports differentiation of cultured osteoblast-like cells. The current study was undertaken to determine whether expression patterns of tenascin-C in TGF-?-treated bone cells are in agreement with a role for this protein

E. J. Mackie; L. A. Abraham; S. L. Taylor; R. P. Tucker; L. I. Murphy

1998-01-01

238

High density cell culture system  

NASA Technical Reports Server (NTRS)

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

Spaulding, Glenn F. (inventor)

1994-01-01

239

NK cells in allogeneic bone marrow transplantation  

Microsoft Academic Search

NK cells, until recently an ignored subset of lymphocytes, have begun to emerge as important cytotoxic effectors. It is now accepted that NK cells together with T cells constitute major actors in graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Over the last several years the mechanisms regulating the activation of NK cells have been the subject of intense investigations

Ioannis A. Voutsadakis

2003-01-01

240

Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Neurocytes and Adipocytes Induced in vitro  

Microsoft Academic Search

Objective) To establish a system for isolation and culture of bone marrow mesenchymal stem cells (MSCs) from Kun-ming mouse in intro and to identify the characteristics of the cells, directional neuronal and adipocytic induction after culture expansion. (Method)MSCs were isolated and cultivated from the bone marrow of Kun-ming mice by investigating their adherence-dependent growth characters. The morphological characters of MSCs

ZHAO Xing; BAI Xue-jin; FENG Ji-wu; DONG Ya-juan

2008-01-01

241

Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells into Neurocytes and Adipocytes Induced in vitro  

Microsoft Academic Search

Objective) To establish a system for isolation and culture of bone marrow mesenchymal stem cells (MSCs) from Kun-ming mouse in intro and to identify the characteristics of the cells, directional neuronal and adipocytic induction after culture expansion. (Method)MSCs were isolated and cultivated from the bone marrow of Kun-ming mice by investigating their adherence-dependent growth characters. The morphological characters of MSCs

242

Netrin-4 derived from murine vascular endothelial cells inhibits osteoclast differentiation in vitro and prevents bone loss in vivo.  

PubMed

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin-4 inhibits osteoclast differentiation in vitro and in vivo. Co-cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin-4, restored in vitro osteoclast differentiation. Administration of netrin-4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin-4. PMID:24846137

Enoki, Yuichiro; Sato, Tsuyoshi; Tanaka, Shinya; Iwata, Takanori; Usui, Michihiko; Takeda, Shu; Kokabu, Shoichiro; Matsumoto, Masahito; Okubo, Masahiko; Nakashima, Keisuke; Yamato, Masayuki; Okano, Teruo; Fukuda, Toru; Chida, Dai; Imai, Yuuki; Yasuda, Hisataka; Nishihara, Tatsuji; Akita, Masumi; Oda, Hiromi; Okazaki, Yasushi; Suda, Tatsuo; Yoda, Tetsuya

2014-06-27

243

Fluid Flow Induced Calcium Response in Bone Cell Network  

PubMed Central

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95–107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18?-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE2 or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18?-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

Huo, Bo; Lu, Xin L.; Hung, Clark T.; Costa, Kevin D.; Xu, Qiaobing; Whitesides, George M.; Guo, X. Edward

2010-01-01

244

Adult rat and human bone marrow stromal cells differentiate into neurons  

Microsoft Academic Search

Bone marrow stromal cells exhibit multiple traits of a stem cell population. They can be greatly expanded in vitro and induced to differentiate into multiple mesenchymal cell types. However, differentiation to non-mesenchymal fates has not been demonstrated. Here, adult rat stromal cells were expanded as undifferentiated cells in culture for more than 20 passages, indicating their proliferative capacity. A simple

Dale Woodbury; Emily J. Schwarz; Darwin J. Prockop; Ira B. Black

2000-01-01

245

Incorporation of Bone Marrow Cells in Pancreatic Pseudoislets Improves Posttransplant Vascularization and Endocrine Function  

PubMed Central

Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×103 cells. To create bone marrow cell-enriched pseudoislets 2×103 islet cells were co-cultured with 2×103 bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.

Wittig, Christine; Laschke, Matthias W.; Scheuer, Claudia; Menger, Michael D.

2013-01-01

246

Suspension culture of mammalian cells.  

PubMed

Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this theme will evolve in the near future at both the pilot and production scales. PMID:1367062

Birch, J R; Arathoon, R

1990-01-01

247

Aflatoxins in Mammalian Cell Cultures.  

National Technical Information Service (NTIS)

KB and HeLa mammalian tissue culture cells were cultured in the presence of 0.4, 1, and 4 ppm of aflatoxin. The incorporation of labeled uridine into the different RNA components separated by sucrose gradient ultracentrifugation and by methylated albumin ...

R. A. Chung

1968-01-01

248

Cell Culture Models for Neurotoxicology  

Microsoft Academic Search

A range of in vitro cell culture methods are available for neurotoxicology which typically represent one of the two predominant cell types present in the brain, neurons and glial cells. These systems can be used in a two tiered approach, whereby simple cytotoxic models reveal the gross effects of a drug or compound and, subsequently, more complex and subtle assays

Glyn Stacey; Barbara Viviani

2001-01-01

249

Enhanced Cell Culture Growth Rates.  

National Technical Information Service (NTIS)

Major project tasks included assembly of an ultrasonic treatment array; measurement of the cell culture growth rate as a function of media concentration, ultrasonic frequency, and ultrasonic power level and dosage; and evaluation of some physiological ind...

S. R. Taylor

1988-01-01

250

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

PubMed

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

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

251

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

PubMed Central

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.

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

252

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

SciTech Connect

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

Long, G.J.; Rosen, J.F.; Pounds, J.G. (Brookhaven National Laboratory, Upton, NY (USA))

1990-02-01

253

MESENCHYMAL STEM CELLS AND THEIR PROGENY: DEVELOPMENTAL PARADIGMS GOVERNING OSTEOBLAST DIFFERENTIATION & BONE FORMATION  

Microsoft Academic Search

1 . We will provide insights into the developmental paradigms governing osteoblast differentiation and bone formation from cellular and molecular analyses of developing bone colonies in vitro. METHODS: Cells were isolated from 21 day Wistar rat calvariae, plated at different densities and cultured for up to 3-4 weeks in differentiation medium (?MEM, with antibiotics, 10% FBS, 50 ?g\\/ml ascorbic acid,

Jane E. Aubin; Shulin Zhang; Soshi Uchida

254

Human Alveolar Bone Cells Interact with ProRoot and Tooth-Colored MTA  

Microsoft Academic Search

The cellular response to mineral trioxide aggregate (MTA) is important for the repair and regeneration of periradicular tissues. The purpose of this study was to analyze the response of human alveolar bone cells to MTA. A human alveolar bone chip was obtained from an oral surgical procedure and explant cultures harvested after 3 to 4 weeks of outgrowth in ?-minimum

Ebtehal AL-Rabeah; Hiran Perinpanayagam; Don MacFarland

2006-01-01

255

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

PubMed Central

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

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

2013-01-01

256

Craniosynostosis-associated Fgfr2(C342Y) mutant bone marrow stromal cells exhibit cell autonomous abnormalities in osteoblast differentiation and bone formation.  

PubMed

We recently reported that cranial bones of Fgfr2(C342Y/+) craniosynostotic mice are diminished in density when compared to those of wild type mice, and that cranial bone cells isolated from the mutant mice exhibit inhibited late stage osteoblast differentiation. To provide further support for the idea that craniosynostosis-associated Fgfr mutations lead to cell autonomous defects in osteoblast differentiation and mineralized tissue formation, here we tested bone marrow stromal cells isolated from Fgfr2(C342Y/+) mice for their ability to differentiate into osteoblasts. Additionally, to determine if the low bone mass phenotype of Crouzon syndrome includes the appendicular skeleton, long bones were assessed by micro CT. Fgfr2(C342Y/+) cells showed increased osteoblastic gene expression during early osteoblastic differentiation but decreased expression of alkaline phosphatase mRNA and enzyme activity, and decreased mineralization during later stages of differentiation, when cultured under 2D in vitro conditions. Cells isolated from Fgfr2(C342Y/+) mice also formed less bone when allowed to differentiate in a 3D matrix in vivo. Cortical bone parameters were diminished in long bones of Fgfr2(C342Y/+) mice. These results demonstrate that marrow stromal cells of Fgfr2(C342Y/+) mice have an autonomous defect in osteoblast differentiation and bone mineralization, and that the Fgfr2(C342Y) mutation influences both the axial and appendicular skeletons. PMID:23762837

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

2013-01-01

257

Interactions between breast cancer cells and bone marrow derived cells in vitro define a role for osteopontin in affecting breast cancer cell migration  

Microsoft Academic Search

The preferential metastasis of breast cancer cells to bone is a complex set of events including homing and preferential growth\\u000a which may include unique factors produced by bone cells in the immediate microenvironment. In this study, we evaluated the\\u000a suitability of bone cells derived from orthoplastic surgeries for use in an in vitro co-culture system representing a model\\u000a of the

Konstantin Koro; Stephen Parkin; Brant Pohorelic; An-Dao Yang; Aru Narendran; Cay Egan; Anthony Magliocco

2011-01-01

258

Generation of a Large Number of Connective Tissue Type Mast Cells by Culture of Murine Fetal Skin Cells  

Microsoft Academic Search

We describe a novel culture system for generating large numbers of murine skin-associated mast cells and distinguish their characteristics from bone marrow-derived cultured mast cells. Culture of day 16 fetal skin single cell suspensions in the presence of interleukin-3 and stem cell factor allowed expansion and maturation of mast cells in the presence of stromal cells. The average yield of

Nobuo Yamada; Hironori Matsushima; Yutaka Tagaya; Shinji Shimada; Stephen I. Katz

2003-01-01

259

Murine Bone Marrow Cells Cultured Ex Vivo in the Presence of Multiple Cytokine Combinations Lose Radioprotective and Long-Term Engraftment Potential  

Microsoft Academic Search

The desire to improve engraftment following transplantation of limited numbers of hematopoietic stem cells (HSC) has spurred the investigation of ex vivo stem cell expansion techniques. While sur- rogate outcomes, such as an increase in SCID-repopulating cells, suggest successful stem cell ex- pansion in some studies, it is not clear that such assays predict outcomes using a more clinically rel-

A. Von Drygalski; G. Alespeiti; L. Ren; J. W. Adamson

2004-01-01

260

Establishment of bone marrow and hematopoietic niches in vivo by reversion of chondrocyte differentiation of human bone marrow stromal cells.  

PubMed

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") can establish the hematopoietic microenvironment within heterotopic ossicles generated by transplantation at non-skeletal sites. Here we show that non-mineralized cartilage pellets formed by hBMSCs ex vivo generate complete ossicles upon heterotopic transplantation in the absence of exogenous scaffolds. These ossicles display a remarkable degree of architectural fidelity, showing that an exogenous conductive scaffold is not an absolute requirement for bone formation by transplanted BMSCs. Marrow cavities within the ossicles include erythroid, myeloid and granulopoietic lineages, clonogenic hematopoietic progenitors and phenotypic HSCs, indicating that complete stem cell niches and hematopoiesis are established. hBMSCs (CD146(+) adventitial reticular cells) are established in the heterotopic chimeric bone marrow through a unique process of endochondral bone marrow formation, distinct from physiological endochondral bone formation. In this process, chondrocytes remain viable and proliferate within the pellet, are released from cartilage, and convert into bone marrow stromal cells. Once explanted in secondary culture, these cells retain phenotype and properties of skeletal stem cells ("MSCs"), including the ability to form secondary cartilage pellets and secondary ossicles upon serial transplantation. Ex vivo, hBMSCs initially induced to form cartilage pellets can be reestablished in adherent culture and can modulate gene expression between cartilage and stromal cell phenotypes. These data show that so-called "cartilage differentiation" of BMSCs in vitro is a reversible phenomenon, which is actually reverted, in vivo, to the effect of generating stromal cells supporting the homing of hematopoietic stem cells and progenitors. PMID:24675053

Serafini, Marta; Sacchetti, Benedetto; Pievani, Alice; Redaelli, Daniela; Remoli, Cristina; Biondi, Andrea; Riminucci, Mara; Bianco, Paolo

2014-05-01

261

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

Microsoft Academic Search

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

A. Scutt; E. M. Williamson

2007-01-01

262

Bone marrow cells regenerate infarcted myocardium.  

PubMed

Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease. PMID:11287958

Orlic, D; Kajstura, J; Chimenti, S; Jakoniuk, I; Anderson, S M; Li, B; Pickel, J; McKay, R; Nadal-Ginard, B; Bodine, D M; Leri, A; Anversa, P

2001-04-01

263

Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study.  

PubMed

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79?, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source. PMID:23996194

Pekovits, Karin; Kröpfl, Julia Maria; Stelzer, Ingeborg; Payer, Michael; Hutter, Heinz; Dohr, Gottfried

2013-12-01

264

The Effect of Deproteinized Bovine Bone Mineral on Saos-2 Cell Proliferation  

PubMed Central

Introduction Deproteinized bovine bone mineral (Bio-Oss) is a xenogenic bone substitute, widely used in maxillofacial bone regeneration. The aim of this in vitro study was to investigate its influence on the growth behavior of human osteosarcoma cell line, Saos-2 culture, and compare it with the physiologic dose of Dexamethasone, an inductive factor for osteoblasts. Materials and Methods Human osteosarcoma cells, Saos-2, were cultured on Bio-Oss and their growth rate was compared to Saos-2 cultures treated with Dexamethasone 10-7 M in contrast to cells cultivated in PBS, in the control group. Assessment of proliferation was performed after 24, 36, and 48 hours by counting cells using trypan blue exclusion method. Alkaline phosphatase was measured spectrophotometrically at 405 nm with paranitrophenol buffer. Results After 48 hours, the number of Saos-2 cells increased significantly when subcultured with Bio-Oss. Bio-Oss was more effective on the enhancement of proliferation of Saos-2 cells when compared to the physiologic dose of Dexamethasone (P<0.05). Alkaline phosphatase activity increased in cells grown on Bio-Oss and dexamethasone 10-7 M in contrast to cells cultivated in PBS control group. The greatest level of activity was observed in the group containing Bio-Oss after 48 hour. Conclusion The significant increase of cell proliferation and alkaline phosphatase activity in cells cultured on Bio-Oss, compared to Dexamethasone-treated cells, suggests the important role of this bone substitute in promoting bone regeneration.

Khojasteh, Arash; Ghahremani, Mohammad Hossein; Ostad, Seyed Nasser; Eslami, Mohammad; Motahhary, Pourya; Morad, Golnaz; Shidfar, Shireen

2013-01-01

265

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

PubMed Central

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

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

2012-01-01

266

Chlorophyllous totipotent maize cell cultures  

US Patent & Trademark Office Database

The subject invention provides totipotent, chlorophyllous, cell cultures of maize. In addition, the methods of producing such cultures are applicable to other related species, including cereals such as rice, oats, barley, and heat. The subject cultures are valuable for herbicide studies, studies for enhancing photosynthesis, and genetic manipulation, such as plastid transformation. The methods of the subject invention are capable of providing high percentages of totipotent cells. These cells are capable of sustained cell division and are competent for regeneration over long periods; they provide high-quality target tissue for nuclear and organelle transformation. The invention also describes methods for the introduction of heterologous DNA into the chloroplast genome. The present invention also provides methods, vectors, and gene constructs for enhancing expression of a recombinant nucleic acid sequence in transgenic plants and plant tissues.

2011-10-18

267

Bone cell senescence: mechanisms and perspectives.  

PubMed

Age-related bone loss is in large part the consequence of senescence mechanisms that impact bone cell number and function. In recent years, progress has been made in the understanding of the molecular mechanisms underlying bone cell senescence that contributes to the alteration of skeletal integrity during aging. These mechanisms can be classified as intrinsic senescence processes, alterations in endogenous anabolic factors, and changes in local support. Intrinsic senescence mechanisms cause cellular dysfunctions that are not tissue specific and include telomere shortening, accumulation of oxidative damage, impaired DNA repair, and altered epigenetic mechanisms regulating gene transcription. Aging mechanisms that are more relevant to the bone microenvironment include alterations in the expression and signaling of local growth factors and altered intercellular communications. This review provides an integrated overview of the current concepts and interacting mechanisms underlying bone cell senescence during aging and how they could be targeted to reduce the negative impact of senescence in the aging skeleton. © 2014 American Society for Bone and Mineral Research. PMID:24496911

Marie, Pierre J

2014-06-01

268

Bone Marrow Mesenchymal Stem Cells Provide an Alternate Pathway of Osteoclast Activation and Bone Destruction by Cancer Cells  

Microsoft Academic Search

The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells. This

Yasuyoshi Sohara; Hiroyuki Shimada; Cedric Minkin; Anat Erdreich-Epstein; Jan A. Nolta; Yves A. DeClerck

2005-01-01

269

Bone regeneration with mesenchymal stem cells  

PubMed Central

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

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

2012-01-01

270

Preliminary study on biological properties of adult human bone marrow-derived mesenchymal stem cells  

Microsoft Academic Search

Objective: To establish a method of culture and expansion of adult human bone marrow-derived MSCs in vitro and to explore their biological properties. Methods: Mononuclear cells were obtained from 5 mL adult human bone marrow by\\u000a density gradient centrifugation with Percoll solution. Adult human MSCs were cultured in Dulbecco’s Modified Eagle’s Medium\\u000a with low glucose (LG-DMEM) containing 10% fetal calf

Tao Wu; Hai Bai; Jingchang Wang; Jingyun Shi; Cunbang Wang; Jihong Lu; Jianfeng Ou; Qian Wang

2006-01-01

271

Ketamine is toxic to chondrocyte cell cultures.  

PubMed

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes. We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic. We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain. Cite this article: Bone Joint J 2014; 96-B:989-94. PMID:24986956

Ozturk, A M; Ergun, M A; Demir, T; Gungor, I; Yilmaz, A; Kaya, K

2014-07-01

272

Stem cell-derived endochondral cartilage stimulates bone healing by tissue transformation.  

PubMed

Although bone has great capacity for repair, there are a number of clinical situations (fracture non-unions, spinal fusions, revision arthroplasty, segmental defects) in which auto- or allografts attempt to augment bone regeneration by promoting osteogenesis. Critical failures associated with current grafting therapies include osteonecrosis and limited integration between graft and host tissue. We speculated that the underlying problem with current bone grafting techniques is that they promote bone regeneration through direct osteogenesis. Here we hypothesized that using cartilage to promote endochondral bone regeneration would leverage normal developmental and repair sequences to produce a well-vascularized regenerate that integrates with the host tissue. In this study, we use a translational murine model of a segmental tibia defect to test the clinical utility of bone regeneration from a cartilage graft. We further test the mechanism by which cartilage promotes bone regeneration using in vivo lineage tracing and in vitro culture experiments. Our data show that cartilage grafts support regeneration of a vascularized and integrated bone tissue in vivo, and subsequently propose a translational tissue engineering platform using chondrogenesis of mesenchymal stem cells (MSCs). Interestingly, lineage tracing experiments show the regenerate was graft derived, suggesting transformation of the chondrocytes into bone. In vitro culture data show that cartilage explants mineralize with the addition of bone morphogenetic protein (BMP) or by exposure to human vascular endothelial cell (HUVEC)-conditioned medium, indicating that endothelial cells directly promote ossification. This study provides preclinical data for endochondral bone repair that has potential to significantly improve patient outcomes in a variety of musculoskeletal diseases and injuries. Further, in contrast to the dogmatic view that hypertrophic chondrocytes undergo apoptosis before bone formation, our data suggest cartilage can transform into bone by activating the pluripotent transcription factor Oct4A. Together these data represent a paradigm shift describing the mechanism of endochondral bone repair and open the door for novel regenerative strategies based on improved biology. © 2014 American Society for Bone and Mineral Research. PMID:24259230

Bahney, Chelsea S; Hu, Diane P; Taylor, Aaron J; Ferro, Federico; Britz, Hayley M; Hallgrimsson, Benedikt; Johnstone, Brian; Miclau, Theodore; Marcucio, Ralph S

2014-05-01

273

Bone regeneration using coculture of mesenchymal stem cells and angiogenic cells  

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

274

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen  

PubMed Central

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 and the opportunities for the use of this model in development of therapeutic strategies.

Gordon, Jennifer; Sariyer, Ilker K.; De La Fuente-Granada, Marisol; Augelli, Brian J.; Otte, Jessica; Azizi, S. Ausim; Amini, Shohreh; Khalili, Kamel; Krynska, Barbara

2013-01-01

275

Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells.  

PubMed

To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients. PMID:14674026

Wang, Jin-fu; Wu, Yi-fan; Harrintong, Jenny; McNiece, Ian K

2004-02-01

276

Direct Proliferative Actions of Stem Cell Factor on Murine Bone Marrow Cells in vitro: Effects of Combination with Colony-Stimulating Factors  

Microsoft Academic Search

Stem cell factor (SCF), the ligand for the c-kit protooncogene product, was able to stimulate blast cell and granulocytic colony formation by precursors from normal murine bone marrow. The blast cell colonies contained a high content of progenitor cells able to form macrophage and\\/or granulocyte colonies. Clone transfer studies, the secondary culture of colony cells, and the culture of populations

D. Metcalf; N. A. Nicola

1991-01-01

277

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

Microsoft Academic Search

There is frequently a loss of vertebral bone due to disease or aging. Nacre (mother of pearl from the oyster Pinctada maxima) stimulates bone cell differentiation and bone formation in vitro and in vivo. Experimental bone defects were prepared in the vertebrae of sheep and used to test the suitability of nacre as an injectable osteogenic biomaterial for treating vertebral

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

1999-01-01

278

Mesenchymal Stem Cells in Bone Tissue Regeneration and Application to Bone Healing  

Microsoft Academic Search

This synoptic study gives a concise overview of current knowledge of bone healing, the role of mesenchymal stem cells in bone tissue regeneration and contemporary possibilities of supporting regeneration of damaged bone. Attention of research concerning the healing of fractures with extensive loss of bone tissue following trauma, the treatment of belatedly healing or non-healing fractures or the healing of

Michal Crha; Alois Ne?as; Robert Srnec; Jan Janovec; Ladislav Stehlík; Petr Raušer; Lucie Urbanová; Ladislav Plánka; Josef Jan?á?; Evžen Amler

2009-01-01

279

Cultured Human Renal Cortical Cells  

NASA Technical Reports Server (NTRS)

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

1998-01-01

280

Bone matrix constituents stimulate interleukin-1 release from human blood mononuclear cells.  

PubMed Central

To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis. Images

Pacifici, R; Carano, A; Santoro, S A; Rifas, L; Jeffrey, J J; Malone, J D; McCracken, R; Avioli, L V

1991-01-01

281

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold.  

PubMed

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer-irrigator-aspirator (RIA)-harvested autograft bone was co-cultured with commercially available Orthoss granules. Native MSCs attached to Orthoss were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co-cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45(+) hematopoietic lineage cells and highly proliferative CD90(+) CD73(+) CD105(+) MSCs preferentially colonized Orthoss granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. PMID:23868185

Kouroupis, Dimitrios; Baboolal, Thomas G; Jones, Elena; Giannoudis, Peter V

2013-12-01

282

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

PubMed Central

The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I ?1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

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

2012-01-01

283

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

PubMed

The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I ?1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials. PMID:22447716

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

2012-07-01

284

Candidates Cell Sources to Regenerate Alveolar Bone from Oral Tissue  

PubMed Central

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

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

2012-01-01

285

CpG oligodeoxynucleotide induces bone marrow precursor cells into myeloid-derived suppressor cells.  

PubMed

Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) perform a number of functions in different immunological settings. In standard in vitro experiments, DCs are produced from mouse bone marrow (BM) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Our previous study demonstrated that BM precursor cells could differentiate into MDSCs when co-cultured with poly (I:C). In the present study, BM precursor cells cultured in GM-CSF and IL-4 were treated with CpG oligodeoxynucleotide (CpG ODN). We observed that Gr1+CD11b+ cells exhibiting MDSC functions accumulated in the co-culture system. A similar phenomenon was also observed in Listeria monocytogenes-infected mice. In conclusion, we demonstrated that prolonged CpG ODN stimulation could inhibit the development of DCs and induce the differentiation of BM precursor cells into MDSCs. PMID:23982226

Chen, Jie; Deng, Chengyu; Shi, Qingmin; Jiang, Jun; Zhang, Yongbo; Shan, Wei; Sun, Weimin

2013-10-01

286

Identification of a hypoxic population of bone marrow cells  

SciTech Connect

A technique using collagenase has been devised to release and separate, with reproducibility, hematopoietic cells (HC) from various microenvironments of mouse femurs. HC were assayed by an in vitro gel culture technique used traditionally to score granulocyte-macrophage precursor cells (CFU-C). CFU-C which resided in the medullary cavity and endosteal regions were sensitive to ionizing radiation and resistant to misonidazole (MISO) cytotoxicity. CFU-C which resided within the compact bone were resistant to ionizing radiation and sensitive to the cytotoxic action of MISO. These results suggest that HC which reside in the bone are hypoxic and retain clonogenic potential. When animals were exposed to various treatments with MISO followed by myelotoxic doses of cyclophosphamide (CTX) or total body irradiation (TBI), the LD/sub 50/ of both agents was significantly reduced. This result suggests that a hypoxic component of HC could be important in the regenerative process within the marrow after such myelotoxic trauma.

Allalunis, M.J.; Chapman, J.D.; Turner, A.R.

1983-02-01

287

Engineering tubular bone using mesenchymal stem cell sheets and coral particles  

SciTech Connect

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.

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

2013-04-19

288

Behaviour of human osteoblastic cells cultured on plasma-sprayed titanium implants in the presence of nicotine  

Microsoft Academic Search

Objectives: The aim of this work was to analyse the behaviour of human bone marrow osteoblastic cells cultured on the surface of routinely used plasma-sprayed titanium implants in the presence of plasmatic and salivary nicotine levels reported in smokers. Material and methods: Human bone marrow cells (first subculture) were seeded on titanium implants and cultured for 35 days in a-minimal

Maria Lurdes Pereira; João Costa Carvalho; Fernando Peres; Manuel Gutierres; Maria Helena Fernandes

2008-01-01

289

Human saliva exposure modulates bone cell performance in vitro.  

PubMed

Various situations encountered by a clinician during the daily routine including surgical periodontitis therapy, dental implant insertion, or tooth extraction involve the contact of saliva with the jaw bone. However, there are only sparse data concerning the influence of saliva on bone cells. Saliva specimens were incorporated within culture medium and administered to murine MC3T3 osteoblasts, of which the morphology (REM), proliferation (EZ4U), and differentiation (qRT-PCR, alkaline phosphatase activity, extracellular matrix calcification) were assessed. Simultaneously, the composition of saliva media was analyzed with respect to the content of lactoferrin, activities of classical salivary enzymes, and the ability to provoke inflammatory cytokine production (enzyme-linked immunosorbent assay) in MC3T3 osteoblasts. The morphology, proliferation, and expression of differentiation-associated genes were seriously handicapped by saliva contact. Saliva-touched cells exhibited less alkaline phosphatase but normal levels of extracellular matrix mineralization. Saliva-containing culture media featured physiological activities of salivary enzymes and considerable amounts of lactoferrin but almost completely lacked salivary alkaline phosphatase and unspecific proteases. Upon saliva incubation, MC3T3 osteoblasts did not release noteworthy levels of interleukin-1 beta or tumor necrosis factor alpha. Although saliva is generally considered to vitalize oral tissues, this study reveals that it harms osteoblast-like cells more due to the presence of salivary enzymes than by triggering of inflammation. This issue is clinically relevant because it broadens the understanding of the bone cell fate within the rather complex cosmos of the oral cavity thereby providing a basis for clinical decision making and treatment guidelines. It seems to be reasonable to restrict the contact period between saliva and bone. PMID:21246386

Proksch, Susanne; Steinberg, Thorsten; Keller, Constantin; Wolkewitz, Martin; Wiedmann-Al-Ahmad, Margit; Finkenzeller, Guenter; Hannig, Christian; Hellwig, Elmar; Al-Ahmad, Ali

2012-02-01

290

Treatment of severe post-traumatic bone defects with autologous stem cells loaded on allogeneic scaffolds.  

PubMed

Mesenchymal stem cells may differentiate into angiogenic and osteoprogenitor cells. The effectiveness of autologous pluripotent mesenchymal cells for treating bone defects has not been investigated in humans. We present a case series to evaluate the rationale of using nucleated cells from autologous bone marrow aspirates in the treatment of severe bone defects that failed to respond to traditional treatments. Ten adult patients (mean age, 49.6-years-old) with severe bone defects were included in this study. Lower limb bone defects were >or=5 cm3 in size, and upper limb defects .or=2 cm3. Before surgery, patients were tested for antibodies to common pathogens. Treatment consisted of bone allogeneic scaffold enriched with bone marrow nucleated cells harvested from the iliac crest and concentrated using an FDA-approved device. Postsurgery clinical and radiographic follow-up was performed at 1, 3, 6, and 12 months. To assess viability, morphology, and immunophenotype, bone marrow nucleated cells were cultured in vitro, tested for sterility, and assayed for the possible replication of adventitious (contaminating) viruses. In 9 of 10 patients, both clinical and radiographic healing of the bone defect along with bone graft integration were observed (mean time, 5.6 months); one patient failed to respond. No post-operative complications were observed. Bone marrow nucleated cells were enriched 4.49-fold by a single concentration step, and these enriched cells were free of microbial contamination. The immunophenotype of adherent cells was compatible with that of mesenchymal stem cells. We detected the replication of Epstein-Barr virus in 2/10 bone marrow cell cultures tested. Hepatitis B virus, cytomegalovirus, parvovirus B19, and endogenous retrovirus HERV-K replication were not detected. Overall, 470 to 1,150 million nucleated cells were grafted into each patient. This case series, with a mean follow-up of almost 2 years, demonstrates that an allogeneic bone scaffold enriched with concentrated autologous bone marrow cells obtained from the iliac crest provides orthopedic surgeons a novel option for treating important bone defects that are unresponsive to traditional therapies. PMID:23065806

Vulcano, Ettore; Murena, Luigi; Cherubino, Paolo; Falvo, Daniele A; Rossi, Antonio; Baj, Andreina; Toniolo, Antonio

2012-12-01

291

Generation of Valpha14 NKT cells in vitro from hematopoietic precursors residing in bone marrow and peripheral blood.  

PubMed

We previously reported the generation of Valpha14 invariant TCR+ (Valpha14i) NK1.1+ natural killer T (NKT) cells in the cytokine-activated suspension culture of murine fetal liver cells. In this study, we attempted to apply this finding to the induction of Valpha14i NKT cell differentiation in the culture of hematopoietic precursors residing in bone marrow or peripheral blood. Preferential generation of NKT cells was found in the culture of Thy-1(+)-depleted bone marrow cells in the presence of culture supernatant from Con A-stimulated spleen T cells and a combination of recombinant IL-3, IL-4, IL-7 and GM-CSF. NKT cell development from peripheral blood hematopoietic precursors was induced when they were cultured on stromal cell monolayers prepared from Thy-1(+)-depleted bone marrow or fetal liver cells, suggesting that certain environments derived from hematopoietic organs are required for the induction of NKT cells from precursors in vitro. A significant fraction of NKT cells generated in the culture were positive for staining with CD1-alpha-galactosylceramide tetramer, indicating that Valpha14i NKT cells were the major subset among the NKT cells. The present methods for obtaining NKT cells in the culture of bone marrow or peripheral blood cells are applicable to the treatment of patients suffering from diseases with numerical and functional disorders of NKT cells. PMID:14991603

Shimamura, Michio; Kobayashi, Kumi; Watanabe, Hiroko; Huang, Yi-Ying; Okamoto, Naoki; Kanie, Osamu; Goji, Hiroshi; Kobayashi, Masumi

2004-03-01

292

Cell Biology of Thiazide Bone Effects  

NASA Astrophysics Data System (ADS)

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

Gamba, Gerardo; Riccardi, Daniela

2008-09-01

293

Identification of Ror? targets in cultured osteoblasts and in human bone  

SciTech Connect

Highlights: •We examine the gene expression patterns controlled by Ror? in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Ror? mRNA levels increase in aged, human bone biopsies. •Ror? 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 ? (Ror?) 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 Ror? is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Ror? are unknown. Using microarray analysis, we identified 281 genes regulated by Ror? in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Ror?-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 Ror?-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Ror?-GFP model system, suggesting that Ror? may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both ROR? and a subset of ROR?-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 ROR? in human age-related bone loss. Collectively, these data demonstrate that Ror? regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

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

294

Matrix metalloproteinase regulation of sphingosine-1-phosphate-induced angiogenic properties of bone marrow stromal cells  

Microsoft Academic Search

Objective. Bone marrow–derived stromal cells (MSC) are able to acquire histological and immunophenotypic characteristics consistent with endothelial cells (EC). In this study we examined the effect of sphingosine-1-phosphate (S1P), a platelet-derived bioactive lysophospholipid that is believed to specifically stimulate EC migration and tube formation, on the angiogenic properties of MSC.Methods. MSC were isolated from murine bone marrow and cultured in

Borhane Annabi; Sébastien Thibeault; Ying-Ta Lee; Nathalie Bousquet-Gagnon; Nicoletta Eliopoulos; Stéphane Barrette; Jacques Galipeau; Richard Béliveau

2003-01-01

295

Bioactive fatty acids: role in bone biology and bone cell function  

Microsoft Academic Search

Bone is a unique tissue providing support, movement, and mineral balance for the body. Bone growth is achieved in the young by a process called modeling, and maintained during adulthood by a process termed remodeling. Three types of cells are responsible for the formation of cartilage and bone; the chondrocyte, osteoblast, and osteoclast. These cells are under the influence of

B. A Watkins; H. E Lippman; L Le Bouteiller; Y Li; M. F Seifert

2001-01-01

296

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines.  

PubMed

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34+ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34+ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5-50 ng/ml had not effect on the expansion of CD34+ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34+ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3. PMID:18029192

Grassinger, Jochen; Simon, Michaela; Mueller, Gunnar; Drewel, Diana; Andreesen, Reinhard; Hennemann, Burkhard

2007-12-01

297

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

PubMed

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

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

2013-12-01

298

Breast cancer micrometastases: different interactions of carcinoma cells with normal and cancer patients' bone marrow stromata.  

PubMed

The apparently dormant breast cancer micrometastases in haemopoietic marrow are correlated with distant metastatic carcinoma dissemination. We studied in vitro interactions of carcinoma cells with adjacent stromata, using connective tissue cell cultures from breast and bone marrow samples of normal donors, comparing them to the pericancerous breast tissue and bone marrows of 12 selected patients with invasive breast carcinomas. Cancer cells were detected by immunocytochemistry and RT-PCR in all the bone marrows and in most blood samples of the studied patients. We monitored the growth and interaction of carcinoma MCF-7 cells with the stromata. The normal breast stroma sustained typical massive cancer growth. The pericancerous breast stroma induced the invasive mesenchymal pattern of growth. Normal bone marrow stroma induced the same conversion and was highly adhesive, retaining the cells in the stroma, but carcinoma patients' bone marrow stromata underwent low adhesive interactions with cancer cells, releasing them potentially into the circulation. The semi-quantitative RT-PCR indicated an enhanced expression of the hepatocyte growth factor and its receptor c-met in breast and bone marrow stromata of cancer patients. The input of cancer cells into the normal bone marrow may induce modifications of the local microenvironment, favourable for growth and release of carcinoma cells into the systemic circulation, which correlate with the poor prognosis of patients with bone marrow micrometastases. PMID:14524537

Nicola, Maria-Helena A; Bizon, Rosana; Machado, Janaina J S; Sollero, Tereza; Rodarte, Renato S; Nobre, João S; Magalhães, Maurício M; Takiya, Christina M; Borojevic, Radovan

2003-01-01

299

The effects of simulated hypogravity on murine bone marrow cells  

NASA Technical Reports Server (NTRS)

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

Lawless, Desales

1989-01-01

300

Different effects on bone strength and cell differentiation in pre pubertal caloric restriction versus hypothalamic suppression?,??  

PubMed Central

Hypothalamic amenorrhea and energy restriction during puberty affect peak bone mass accrual. One hypothesis suggests energy restriction alters hypothalamic function resulting in suppressed estradiol levels leading to bone loss. However, both positive and negative results have been reported regarding energy restriction and bone strength. Therefore, the purpose of this study was to investigate energy restriction and hypothalamic suppression during pubertal onset on bone mechanical strength and the osteogenic capacity of bone marrow-derived cells in two models: female rats treated with gonadotropin releasing hormone antagonists (GnRH-a) or 30% energy restriction. At 23 days of age, female Sprague Dawley rats were assigned to three groups: control group (C, n=10), GnRH-a group (n=10), and Energy Restriction (ER, n=12) group. GnRH-a animals received daily injections for 27 days. The animals in the ER group received 70% of the control animals’ intake. After sacrifice (50 days of age), body weight, uterine and muscle weights were measured. Bone marrow-derived stromal cells were cultured and assayed for proliferation and differentiation into osteoblasts. Outcome measures included bone strength, bone histomorphometry and architecture, serum IGF-1 and osteocalcin. GnRH-a suppressed uterine weight, decreased osteoblast proliferation, bone strength, trabecular bone volume and architecture compared to control. Elevated serum IGF-1 and osteocalcin levels and body weight were found. The ER model had an increase in osteoblast proliferation compared to the GnRH-a group, similar bone strength relative to body weight and increased trabecular bone volume in the lumbar spine compared to control. The ER animals were smaller but had developed bone strength sufficient for their size. In contrast, suppressed estradiol via hypothalamic suppression resulted in bone strength deficits and trabecular bone volume loss. In summary, our results support the hypothesis that during periods of nutritional stress the increased vertebral bone volume may be an adaptive mechanism to store mineral which differs from suppressed estradiol resulting from hypothalamic suppression.

Joshi, R.N.; Safadi, F.F.; Barbe, M.F.; Carpio-Cano, Fe Del; Popoff, S.N.; Yingling, V.R.

2013-01-01

301

Evaluation of critical size defects of mouse calvarial bone: An organ culture study.  

PubMed

Mouse calvarial organ culture has been used widely for the study of bone biology. The purpose of this study was to evaluate the healing potential of neonatal mouse parietal defects in different culture media. The critical size defect (CSD) was also investigated. The parietal bones of neonatal mice were used. Full-thickness, 0.8-mm circular defects were created through the bones from one litter of mice. The bones were divided into three groups: Dulbecco's Modified Eagle Medium (DMEM) group, DMEM/osteogenic medium (OM) group, and OM group. Cultures were analyzed with microcomputed-tomography, dissecting-microscope, phase-contrast-microscope, Von Kossa stain, scanning-electron-microscopy, and energy-dispersive-X-ray. Continuous bone formation of parietal bones was observed in all groups. Defects in the DMEM/OM group showed the highest healing potential and exhibited woven bone formation. Defects in the OM group showed limited bone healing at the defect edge. Defects in the DMEM group showed fibrous healing. The most effective culture medium (DMEM/OM) was used to determine the CSD of mouse calvaria in a separate experiment. Circular defects (diameters: 0.8, 1.0, and 1.5 mm) were made in the parietal bones from another litter of mice. The bones were analyzed with microcomputed-tomography, and phase-contrast-microscopy. The bone filling percentages of different size defects were statistically significant: 1.5-mm defects (4.49%), 1.0-mm defects (47.65%), and 0.8-mm defects (73.45%). In three culture conditions, DMEM/OM was the most effective approach to repair bone defects. A 1.5 mm in diameter, full-thickness parietal defect was found to be the CSD under the DMEM/OM culture conditions. PMID:19937748

Wu, Xiaohong; Downes, Sandra; Watts, David C

2010-05-01

302

Dissecting the Role of Bone Marrow Stromal Cells on Bone Metastases  

PubMed Central

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

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

2014-01-01

303

Cell culture in vivo by means of diffusion chamber system.  

PubMed

In a diffusion chamber (DC) system, cells are cultured in vivo - hence making it possible to minimize infection and foreign material contamination. In view of this merit, we devised a technique to combine a DC system and a scaffold to the end of incubating sufficient host cells for grafting. In the present study, PLGA sponge and rat bone marrow cells were encapsulated inside a DC and then placed inside the abdominal cavities of rats. DCs were removed at two or four weeks after grafting. At four weeks after grafting, fibrous and calcified tissue matching the shape of the PLGA sponge was formed. These results suggested that the PLGA sponge was an effective scaffolding material in inducing three-dimensional tissue formation and that combination with a DC system resulted in a cell mass matching the scaffold shape. In addition, the cells were cultured in vivo - which meant that DC culturing did not require special incubation facilities or technologies after grafting. PMID:19721273

Nakano, Kenjiro; Hayashi, Tatsuhide; Kawai, Hideki; Takei, Yukiko; Sato, Yosuke; Ando, Kimitoshi; Ono, Yuzo; Jinno, Satoshi; Kawakami, Toshiyuki; Maeda, Hatuhiko; Kawai, Tatsushi

2009-07-01

304

Stem cells today: B1. Bone marrow stem cells  

Microsoft Academic Search

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

RG Edwards

2004-01-01

305

Trends in cell culture technology.  

PubMed

Dynamic macroscale bioreactor systems are the most recent breakthrough in cell culture technology. This major achievement, at the beginning of the 21st century, fortunately coincided with an embarrassing gap in the measures to predict the safety and modes of action of chemicals, cosmetics, air particles and pharmaceuticals. The major hurdles to the translation of these breakthrough achievements of cell culture technology into meaningful solutions for predictive high throughput substance testing remain miniaturization from the milliliter to the microliter scale and the supply of relevant amounts of standardized human tissue. This chapter provides insights into the latest developments in this area, illustrates an original multi-micro-organ bioreactor concept and identifies highways for closing the gap. PMID:22437811

Marx, Uwe

2012-01-01

306

Unique cell culture systems for ground based research  

NASA Technical Reports Server (NTRS)

The horizontally rotating fluid-filled, membrane oxygenated bioreactors developed at NASA Johnson for spacecraft applications provide a powerful tool for ground-based research. Three-dimensional aggregates formed by cells cultured on microcarrier beads are useful for study of cell-cell interactions and tissue development. By comparing electron micrographs of plant seedlings germinated during Shuttle flight 61-C and in an earth-based rotating bioreactor it is shown that some effects of microgravity are mimicked. Bioreactors used in the UAH Bioreactor Laboratory will make it possible to determine some of the effects of altered gravity at the cellular level. Bioreactors can be valuable for performing critical, preliminary-to-spaceflight experiments as well as medical investigations such as in vitro tumor cell growth and chemotherapeutic drug response; the enrichment of stem cells from bone marrow; and the effect of altered gravity on bone and muscle cell growth and function and immune response depression.

Lewis, Marian L.

1990-01-01

307

Bone Repair Cells for Craniofacial Regeneration  

PubMed Central

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

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

2012-01-01

308

Engineering bone formation from human dental pulp- and periodontal ligament-derived cells.  

PubMed

A robust method for inducing bone formation from cultured dental mesenchymal cells has not been established. In this study, a method for generating bone tissue in vivo from cultured human dental pulp- and periodontal ligament-derived cells (DPCs and PDLCs, respectively) was designed using exogenous bone morphogenetic protein 2 (BMP2). DPCs and PDLCs showed enhanced alkaline phosphatase (ALP) activity and calcified nodule formation in medium containing dexamethasone, ?-glycerophosphate, and ascorbic acid (osteogenic medium). However, the addition of recombinant human bone morphogenetic protein 2 (rhBMP2) to osteogenic medium remarkably increased ALP activity and in vitro calcification above the increases observed with osteogenic medium alone. rhBMP2 also significantly upregulated the expression of osteocalcin, osteopontin, and dentin matrix protein 1 mRNA in both cell types cultured in osteogenic medium. Finally, we detected prominent bone-like tissue formation in vivo when cells had been exposed to rhBMP2 in osteogenic medium. In contrast, treatments with osteogenic medium or rhBMP2 alone could not induce abundant mineralized tissue formation. We propose here that treatment with rhBMP2 in osteogenic medium can make dental mesenchymal tissues a highly useful source of cells for bone tissue engineering. In addition, both DPCs and PDLCs showed similar and remarkable osteo-inducibility. PMID:20614244

Ikeda, Hideyoshi; Sumita, Yoshinori; Ikeda, Mihoko; Ikeda, Hisazumi; Okumura, Teruhito; Sakai, Eiko; Nishimura, Masahiro; Asahina, Izumi

2011-01-01

309

Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors.  

PubMed

Human bone marrow contains a population of bone marrow stromal cells (hBMSCs) capable of forming several types of mesenchymal tissues, including bone and cartilage. The present study was designed to test whether large cartilaginous and bone-like tissue constructs can be selectively engineered using the same cell population (hBMSCs), the same scaffold type (porous silk) and same hydrodynamic environment (construct settling in rotating bioreactors), by varying the medium composition (chondrogenic vs. osteogenic differentiation factors). The hBMSCs were harvested, expanded and characterized with respect to their differentiation potential and population distribution. Passage two cells were seeded on scaffolds and cultured for 5 weeks in bioreactors using osteogenic, chondrogenic or control medium. The three media yielded constructs with comparable wet weights and compressive moduli ( approximately 25 kPa). Chondrogenic medium yielded constructs with higher amounts of DNA (1.5-fold) and glycosaminoglycans (GAG, 4-fold) per unit wet weight (ww) than control medium. In contrast, osteogenic medium yielded constructs with higher dry weight (1.6-fold), alkaline phosphatase (AP) activity (8-fold) and calcium content (100-fold) per unit ww than control medium. Chondrogenic medium yielded constructs that were weakly positive for GAG by contrast-enhanced MRI and alcian blue stain, whereas osteogenic medium yielded constructs that were highly mineralized by microCT and von Kossa stain. Engineered bone constructs were large (8mm diameter x 2mm thick disks) and resembled trabecular bone with respect to structure and mineralized tissue volume fraction (12%). PMID:16895736

Marolt, Darja; Augst, Alexander; Freed, Lisa E; Vepari, Charu; Fajardo, Robert; Patel, Nipun; Gray, Martha; Farley, Michelle; Kaplan, David; Vunjak-Novakovic, Gordana

2006-12-01

310

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells  

SciTech Connect

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

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

2007-12-31

311

Interleukin 1 synthesis by a transitional cell carcinoma: relationship to bone resorption and humoral hypercalcemia of malignancy  

Microsoft Academic Search

A tumor cell line (TCCB) was isolated from a patient with transitional cell carcinoma, who had tumor-associated hypercalcemia. Culture supernatants from TCCB had significant bone resorption activity in a ⁴⁵Ca release assay from fetal mouse long bones. Furthermore, TCCB supernatants possessed potent interleukin 1 (IL-1) activity in a mouse thymocyte assay. Histomorphometric analysis of fetal mouse long bones incubated with

P. Sammon; T. Wronski; L. Ignaszewski; J. Flueck; D. A. Cohen

1986-01-01

312

Effect of platelet releasate on bone cell migration and recruitment in vitro.  

PubMed

The use of platelets and platelet products has become increasingly popular clinically as a means of accelerating endosseous wound healing. It is likely that growth factors released by activated platelets at the site of injury play a role in bone regeneration by stimulating the migration and proliferation of bone cells. In this study, a novel in vitro assay was developed to study the effects of platelet releasate (PR) collected from activated platelet concentrate on rat bone marrow-derived cells. Cultures of primary rat bone marrow cells were overlaid with a fibrin matrix, and the number of cells migrating within the three-dimensional matrix and the leading front of migration were quantified. The addition of PR to the top of the fibrin gels at different time points caused a 25% increase in the leading front of migration and a 3.5-fold increase in the number of migrating cells. Platelet releasate was also shown to have a mitogenic effect on bone cells in proliferation studies. Comparison between migration and proliferation data indicated that PR stimulates the initial recruitment of bone marrow cells to migration. This assay further allowed the determination that rat bone marrow cells are capable of exerting contractile forces on fibrin matrices and that matrix contraction is directly related to the migratory activity of cells. The results provide a potential mechanism to explain why biologically active platelet-derived factors enhance endosseous wound healing. PMID:12826799

Oprea, Wanda E; Karp, Jeffrey M; Hosseini, Morris M; Davies, John E

2003-05-01

313

Isolation, differentiation, and characterisation of skeletal stem cells from human bone marrow in vitro and in vivo.  

PubMed

In this chapter, we describe techniques for the isolation and characterization of skeletal stem cells from human bone marrow. The methods for enrichment of STRO-1 positive cells using magnetic activated cell sorting are described and we also cover techniques for establishing and characterising osteogenic, adipogenic, and chondrogenic cultures from these cells. Finally, we present methods for studying the ability of these cells to produce bone in vivo using diffusion chambers which have been implanted subcutaneously in mice. PMID:22130924

Tare, Rahul S; Mitchell, Peter D; Kanczler, Janos; Oreffo, Richard O C

2012-01-01

314

Sca1and Thy1 Accelerate Neuron-like Differentiation in Bone Marrow Stromal Cells  

Microsoft Academic Search

Bone marrow stromal cells taken from EGFP transgenic mice were sorted by magnetic beads with surface markers for Sca-1 and Thy-1. The cells were then co-cultured on organotypic hippocampal slice or with neuronal cell feeder in dish. On hippocampus, both Sca-1 and Thy-1 positive cells showed 4- 8 folds higher potential to show neuron-like morphology than negative cells. In dish,

TAKASHI MIZOBE; KEIJI KIDOGUCHI; MASAHIRO TAMAKI; TAKASHI SASAYAMA; TAKESHI KONDOH; EIJI KOHMURA

2006-01-01

315

Chromosome preparation from cultured cells.  

PubMed

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15). PMID:24513647

Howe, Bradley; Umrigar, Ayesha; Tsien, Fern

2014-01-01

316

Human bone-derived cells support formation of human osteoclasts from arthroplasty-derived cells in vitro.  

PubMed

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH)2 vitamin D3. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur. Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH)2 vitamin D3 was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE2. Exogenous PGE2 (10(-8) to 10(-6) M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE2 by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening. PMID:10990320

Neale, S D; Fujikawa, Y; Sabokbar, A; Gundle, R; Murray, D W; Graves, S E; Howie, D W; Athanasou, N A

2000-08-01

317

In vivo alveolar bone regeneration by bone marrow stem cells\\/fibrin glue composition  

Microsoft Academic Search

The repair of alveolar bone defects caused by trauma, periodontal diseases and inflammation is still a challenge for both researchers and clinicians. Although there are many attempts to regenerate bone based on different seed cells and scaffolds, the results are still unsatisfactory. This study aims to clarify whether it could be efficient to reconstruct the alveolar bone by the combination

Liang Zhang; Peihuan Wang; Shenglin Mei; Chenghua Li; Chuan Cai; Yin Ding

318

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

Microsoft Academic Search

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

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

1986-01-01

319

Painful scoliosis due to superposed giant cell bone tumor and aneurysmal bone cyst in a child.  

PubMed

Giant cell bone tumors are the most common precursor lesions of aneurysmal bone cysts (ABCs) developing secondarily. In giant cell bone tumors containing an explicit ABC component, the observation of the solid component of the giant cell bone tumor plays a critical role in the separation of the primary ABC. In general, ABC cases together with giant cell tumors in the bone are diagnosed histopathologically. The combination of giant cell bone tumor with superposed ABC and that of painful scoliosis with backache is rarely seen in children. In this case study, we discussed the diagnosis and the treatment of a giant cell tumor and superposed an ABC present in the fifth lumbar spine in a pediatric patient admitted to our clinic with a complaint of acute scoliotic back pain. PMID:24858183

Togral, Guray; Arikan, Murat; Hasturk, Askin E; Gungor, Safak

2014-07-01

320

Participation of bone marrow derived cells in cutaneous wound healing.  

PubMed

Bone marrow has long been known to be a source of stem cells capable of regeneration of the hematopoeitic system. Recent reports, however, have indicated that bone marrow might also contain early stem cells that can differentiate into other organ tissues such as skin. While these studies have illustrated that bone marrow stem cells could find their way to the skin, they have not addressed the dynamics of how bone marrow stem cells might participate in the homeostatis and regeneration of skin. In this report we followed green fluorescent protein (GFP) labeled bone marrow transplanted into non-GFP mice in order to determine the participation of bone marrow stem cells in cutaneous wounds. Our results indicate that there are a significant number of bone marrow cells that traffic through both wounded and non-wounded skin. Wounding stimulated the engraftment of bone marrow cells to the skin and induced bone marrow derived cells to incorporate into and differentiate into non-hematopoietic skin structures. This report thus illustrates that bone marrow might be a valuable source of stem cells for the skin and possibly other organs. Wounding could be a stimulus for bone marrow derived stem cells to travel to organs and aid in the regeneration of damaged tissue. PMID:12811816

Badiavas, Evangelos V; Abedi, Mehrdad; Butmarc, Janet; Falanga, Vincent; Quesenberry, Peter

2003-08-01

321

Heme oxygenase-1 (HO-1) expression in prostate cancer cells modulates the oxidative response in bone cells.  

PubMed

Prostate cancer (PCa) is a leading cause of death among males. It is currently estimated that inflammatory responses are linked to 15-20% of all deaths from cancer worldwide. PCa is dominated by complications arising from metastasis to the bone where the tumor cells interact with the bone microenvironment impairing the balance between bone formation and degradation. However, the molecular nature of this interaction is not completely understood. Heme oxygenase-1 (HO-1) counteracts oxidative damage and inflammation. Previous studies from our laboratory showed that HO-1 is implicated in PCa, demonstrating that endogenous HO-1 inhibits bone derived-prostate cancer cells proliferation, invasion and migration and decreases tumor growth and angiogenesis in vivo. The aim of this work was to analyze the impact of HO-1 modulated PCa cells on osteoblasts proliferation in vitro and on bone remodeling in vivo. Using a co-culture system of PC3 cells with primary mice osteoblasts (PMOs), we demonstrated that HO-1 pharmacological induction (hemin treatment) abrogated the diminution of PMOs proliferation induced by PCa cells and decreased the expression of osteoclast-modulating factors in osteoblasts. No changes were detected in the expression of genes involved in osteoblasts differentiation. However, co-culture of hemin pre-treated PC3 cells (PC3 Hem) with PMOs provoked an oxidative status and activated FoxO signaling in osteoblasts. The percentage of active osteoblasts positive for HO-1 increased in calvarias explants co-cultured with PC3 Hem cells. Nuclear HO-1 expression was detected in tumors generated by in vivo bone injection of HO-1 stable transfected PC3 (PC3HO-1) cells in the femur of SCID mice. These results suggest that HO-1 has the potential to modify the bone microenvironment impacting on PCa bone metastasis. PMID:24224047

Ferrando, Mercedes; Wan, Xinhai; Meiss, Roberto; Yang, Jun; De Siervi, Adriana; Navone, Nora; Vazquez, Elba

2013-01-01

322

A microfluidic system for automatic cell culture  

NASA Astrophysics Data System (ADS)

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

Huang, Chun-Wei; Lee, Gwo-Bin

2007-07-01

323

Osteoblast-Prostate Cancer Cell Interaction in Prostate Cancer Bone.  

National Technical Information Service (NTIS)

Prostate cancer cells have a remarkable affinity to develop metastases in bone. Clinical data and laboratory observations both suggest that bone-malignant epithelium interactions play a central role in prostate cancer progression. We have developed an in ...

N. M. Navone

2000-01-01

324

Ligand Accumulation in Autocrine Cell Cultures  

Microsoft Academic Search

Cell-culture assays are routinely used to analyze autocrine signaling systems, but quantitative experiments are rarely possible. To enable the quantitative design and analysis of experiments with autocrine cells, we develop a biophysical theory of ligand accumulation in cell-culture assays. Our theory predicts the ligand concentration as a function of time and measurable parameters of autocrine cells and cell-culture experiments. The

Michael I. Monine; Alexander M. Berezhkovskii; Elizabeth J. Joslin; H. Steven Wiley; Douglas A. Lauffenburger; Stanislav Y. Shvartsman

2005-01-01

325

Stimulation of bone resorption in organ culture by salt-free extracts of Solanum glaucophyllum.  

PubMed

Addition of a desalted aqueous extract made from dried leaves of Solanum glaucophyllum produced a marked stimulation of bone resorption in a culture system in vitro. This result indicates that, like the biologically active forms of vitamin D, the active material in the plant not only stimulates intestinal calcium absorption but increases bone mobilization as well. PMID:1140163

Lloyd, W; Wells, H; Walling, M W; Kimberg, D V

1975-01-01

326

Effect of Autologous Bone Marrow Stromal Cell Seeding and Bone Morphogenetic Protein-2 Delivery on Ectopic Bone Formation in a Microsphere/Poly(Propylene Fumarate) Composite  

PubMed Central

A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8??g BMP-2?per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2–loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2–impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8??g/mg BMP-2–loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2–impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.

Kempen, Diederik H.R.; Kruyt, Moyo C.; Lu, Lichun; Wilson, Clayton E.; Florschutz, Anthony V.; Yaszemski, Michael J.; Dhert, Wouter J.A.

2009-01-01

327

The effect of differentiation stage of amniotic fluid stem cells on bone regeneration.  

PubMed

Bone tissue engineering strategies require cells with high proliferative and osteogenic potential as well as a suitable scaffold to support the development of these as they form new bone tissue. In this study, we evaluated whether the differentiation stage of amniotic fluid stem cells (AFSC) could enhance the regeneration of critical sized femoral defects in a rat model. For this purpose, AFSC were seeded onto a starch-poly(?-caprolactone) (SPCL) scaffold and were cultured in vitro in osteogenic culture media for different periods of time in order to obtain: i) undifferentiated cells, ii) cells committed to the osteogenic phenotype and iii) "osteoblast-like" cells. In vitro results indicate that AFSC were considered to be osteogenically committed by the end of week 2 and osteoblastic-like after week 3 in culture. Constructs composed of AFSC-SPCL scaffolds from each differentiation stage were implanted into critical sized femoral defects. The quality of new tissue formed in the defects was evaluated based on micro-CT imaging and histological analysis of constructs retrieved at 4 and 16 weeks after implantation. In vivo formation of new bone was observed under all conditions. However, the most complete repair of the defect was observed after 16 weeks in the animals receiving the SPCL scaffolds seeded with osteogenically committed AFSC. Furthermore, the presence of blood vessels was noted in the inner sections of the scaffolds suggests that these cells could potentially be used to induce bone regeneration and angiogenesis in non-union bone defects. PMID:22672834

Rodrigues, Márcia T; Lee, Bu-Kyu; Lee, Sang Jin; Gomes, Manuela E; Reis, Rui L; Atala, Anthony; Yoo, James J

2012-09-01

328

Developmental Cues for Bone Formation from Parathyroid Hormone and Parathyroid Hormone-Related Protein in an Ex Vivo Organotypic Culture System of Embryonic Chick Femora  

PubMed Central

Enhancement and application of our understanding of skeletal developmental biology is critical to developing tissue engineering approaches to bone repair. We propose that use of the developing embryonic femur as a model to further understand skeletogenesis, and the effects of key differentiation agents, will aid our understanding of the developing bone niche and inform bone reparation. We have used a three-dimensional organotypic culture system of embryonic chick femora to investigate the effects of two key skeletal differentiation agents, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), on bone and cartilage development, using a combination of microcomputed tomography and histological analysis to assess tissue formation and structure, and cellular behavior. Stimulation of embryonic day 11 (E11) organotypic femur cultures with PTH and PTHrP initiated osteogenesis. Bone formation was enhanced, with increased collagen I and STRO-1 expression, and cartilage was reduced, with decreased chondrocyte proliferation, collagen II expression, and glycosaminoglycan levels. This study demonstrates the successful use of organotypic chick femur cultures as a model for bone development, evidenced by the ability of exogenous bioactive molecules to differentially modulate bone and cartilage formation. The organotypic model outlined provides a tool for analyzing key temporal stages of bone and cartilage development, providing a paradigm for translation of bone development to improve scaffolds and skeletal stem cell treatments for skeletal regenerative medicine.

Kanczler, Janos M.; Roberts, Carol A.; Oreffo, Richard O.C.

2012-01-01

329

Developmental cues for bone formation from parathyroid hormone and parathyroid hormone-related protein in an ex vivo organotypic culture system of embryonic chick femora.  

PubMed

Enhancement and application of our understanding of skeletal developmental biology is critical to developing tissue engineering approaches to bone repair. We propose that use of the developing embryonic femur as a model to further understand skeletogenesis, and the effects of key differentiation agents, will aid our understanding of the developing bone niche and inform bone reparation. We have used a three-dimensional organotypic culture system of embryonic chick femora to investigate the effects of two key skeletal differentiation agents, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), on bone and cartilage development, using a combination of microcomputed tomography and histological analysis to assess tissue formation and structure, and cellular behavior. Stimulation of embryonic day 11 (E11) organotypic femur cultures with PTH and PTHrP initiated osteogenesis. Bone formation was enhanced, with increased collagen I and STRO-1 expression, and cartilage was reduced, with decreased chondrocyte proliferation, collagen II expression, and glycosaminoglycan levels. This study demonstrates the successful use of organotypic chick femur cultures as a model for bone development, evidenced by the ability of exogenous bioactive molecules to differentially modulate bone and cartilage formation. The organotypic model outlined provides a tool for analyzing key temporal stages of bone and cartilage development, providing a paradigm for translation of bone development to improve scaffolds and skeletal stem cell treatments for skeletal regenerative medicine. PMID:22690868

Smith, Emma L; Kanczler, Janos M; Roberts, Carol A; Oreffo, Richard O C

2012-12-01

330

Catecholamines in murine bone marrow derived mast cells.  

PubMed

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753+/-844 pg/10(7) cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine < or =10 microM affected IL-3-induced cell proliferation. High exogenous dopamine (20-100 microM) decreased proliferation and increased apoptosis, and the anti-oxidant ascorbic acid prevented these effects. Increased expression of the anti-apoptotic factor Bcl-2 or loss of pro-apoptotic Bax expression attenuated dopamine-induced apoptosis, suggesting the apoptosis proceeds through a mitochondrial pathway. PMID:11585626

Freeman, J G; Ryan, J J; Shelburne, C P; Bailey, D P; Bouton, L A; Narasimhachari, N; Domen, J; Siméon, N; Couderc, F; Stewart, J K

2001-10-01

331

Dynamized Preparations in Cell Culture  

PubMed Central

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

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

2009-01-01

332

LANTHANUM IN HEART CELL CULTURE  

PubMed Central

Correlation of the localization of La+++ with its effects on Ca++ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca++ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca++/kg wet cells or 43% of the exchangeable Ca++ (cells perfused with [Ca++]o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca++/kg wet cells or 57% of the exchangeable Ca++. 0.5 mM [La+++]o displaces 0 52 mmoles Ca++/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca++ influx and efflux The presence of La+++ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca++ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La+++ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle

Langer, G. A.; Frank, J. S.

1972-01-01

333

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

NASA Astrophysics Data System (ADS)

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

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

2000-09-01

334

Skeletal muscle and bone marrow derived stromal cells: a comparison of tenocyte differentiation capabilities.  

PubMed

This study investigated the comparative ability of bone marrow and skeletal muscle derived stromal cells (BMSCs and SMSCs) to express a tenocyte phenotype, and whether this expression could be augmented by growth and differentiation factor-5 (GDF-5). Tissue harvest was performed on the hind limbs of seven dogs. Stromal cells were isolated via serial expansion in culture. After four passages, tenogenesis was induced using either ascorbic acid alone or in conjunction with GDF-5. CD44, tenomodulin, collagen I, and collagen III expression levels were compared for each culture condition at 7 and 14 days following induction. Immunohistochemistry (IHC) was performed to evaluate cell morphology and production of tenomodulin and collagen I. SMSCs and BMSCs were successfully isolated in culture. Following tenocytic induction, SMSCs demonstrated an increased mean relative expression of tenomodulin, collagen I, and collagen III at 14 days. BMSCs only showed increased mean relative expression of collagen I, and collagen III at 14 days. IHC revealed positive staining for tenomodulin and collagen I at 14 days for both cell types. The morphology of skeletal muscle derived stromal cells at 14 days had an organized appearance in contrast to the haphazard arrangement of the bone marrow derived cells. GDF-5 did not affect gene expression, cell staining, or cell morphology significantly. Stromal cells from either bone marrow or skeletal muscle can be induced to increase expression of matrix genes; however, based on expression of tenomodulin and cell culture morphology SMSCs may be a more ideal candidate for tenocytic differentiation. PMID:22511232

Sassoon, Adam A; Ozasa, Yasuhiro; Chikenji, Takako; Sun, Yu-Long; Larson, Dirk R; Maas, Mary L; Zhao, Chunfeng; Jen, Jin; Amadio, Peter C

2012-11-01

335

Experimental model for observation of micromotion in cell culture.  

PubMed

It is known that the micromotion between implant and bone inhibits direct bone growth either on or into implant surfaces in vivo. Nevertheless, biocompatibility tests in vitro of biomaterials for bone/implant interfaces are mainly performed under static conditions. This work describes a dynamic, in vitro experimental simulation of the effect of mutual, small-scale implant surface-tissue displacement on adhered cells. Disks of simulated tissue (PVP hydrogel) were subjected to cyclic micromotion ranging from 0 at the center to 1000 microm at the periphery at approximately 13 Hz, relative to biomaterial surfaces or tissue culture polystyrene controls populated with human osteoblasts in standard tissue culture plate wells. The effect of the interfacial micromotion on the number of cells remaining attached was quantitated by XTT assay. The activity level of the remaining cells was determined by an alkaline phosphatase assay, and cell stress was evaluated by nitrogen assay. Significantly more cells (ANOVA) became detached from similarly prepared surfaces of titanium, hydroxyapatite, and alumina compared to the polystyrene control, and detachment from alumina was greater than for the other two materials. The activity of the remaining attached cells was lower as compared to the static (no micromotion) control but not significantly different among the biomaterials. All nitrogen assays were negative, suggesting minimal cell stress occurred. The method is proposed as a useful and discriminating in vitro tool for biocompatibility studies focused on cell adhesion to biomaterials under conditions related to those which exist at the implant/bone interface in vivo, and it allows subsequent studies of the still-viable cells by other methods. PMID:15654711

Lewandowska-Szumie?, Ma?gorzata; Sikorski, Krzysztof; Szummer, Andrzej; Komender, Janusz; Kowalski, Marcin; Daniels, A U

2005-02-15

336

BMP2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold  

Microsoft Academic Search

Osteogenic differentiation of bone marrow stromal cells (BMSC) in a three-dimensional (3-D) scaffold has not been well studied. In this work, we studied expression of bone-related genes during differentiation of rabbit BMSCs in response to bone morphogenetic protein (BMP)-2 in both 2-D and 3-D culture systems. When BMSCs were cultured on films (2-D) of biodegradable poly(lactide-co-glycolide) (PLGA), increases in mRNA

Weibiao Huang; Brian Carlsen; Isabella Wulur; George Rudkin; Kenji Ishida; Benjamin Wu; Dean T. Yamaguchi; Timothy A. Miller

2004-01-01

337

Injectable Bone Tissue Engineering Using Expanded Mesenchymal Stem Cells  

PubMed Central

Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, very few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue-engineered bone composed of mesenchymal stem cells (MSCs) and platelet rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out pilot trial cases in patients with long-term follow up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months was significantly higher than the pre-operation baseline (P <0.001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long term follow-up. Injectable tissue-engineered bone restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering.

Yamada, Yoichi; Nakamura, Sayaka; Ito, Kenji; Umemura, Eri; Hara, Kenji; Nagasaka, Tetsuro; Abe, Akihiro; Baba, Shunsuke; Furuichi, Yasushi; Izumi, Yuichi; Klein, Ophir D.; Wakabayashi, Toshihiko

2014-01-01

338

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

PubMed

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

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

2010-12-01

339

Culture and differentiation of embryonic stem cells  

Microsoft Academic Search

Summary Techniques are described for the culture of murine embryonic stem cells in the absence of heterologous feeder cells and for the induction of differentiation programs. The regulatory factor differentiation inhibiting activity\\/ leukaemia inhibitory factor (DIA\\/LIF) is produced at high concentration by transient expression in Cos cells and is used to suppress stem cell differentiation by addition to the culture

Austin G. Smith

1991-01-01

340

"Humanized" Stem Cell Culture Techniques: The Animal Serum Controversy  

PubMed Central

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

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

2011-01-01

341

Proliferative activity of vervet monkey bone marrow-derived adherent cells  

SciTech Connect

Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and n = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus, the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

Kramvis, A.; Garnett, H.M.

1987-11-01

342

Cultivation and identification of rat bone marrow?derived mesenchymal stem cells.  

PubMed

Bone marrow?derived mesenchymal stem cells (BMSCs) have the potential to form a variety of mesenchymal tissue types, which are a source of cells for bone tissue engineering applications. The present study attempted to establish an effective and convenient method for culturing BMSCs. Total bone marrow cells, which were harvested from rat femurs, were cultured and BMSCs were selected and expanded through passaging in vitro. Furthermore, the biological properties of BMSCs were investigated, specific surface antigen expression was assessed using flow cytometry and the multipotent differentiation potential characteristics were demonstrated using standard in vitro conditions. Monoptychial heterogeneous cells were obtained. A total of 98.4% of cells at passage 3 expressed cluster of differentiation (CD)29 and CD90, but not CD45. The cells were able to differentiate into osteogenic and adipogenic cells. In conclusion, BMSCs that are isolated from the rat bone marrow and exhibit the identified characteristics may be used as seed cells in bone tissue engineering. PMID:24859847

Song, Ke; Huang, Mengqi; Shi, Qi; Du, Tianfeng; Cao, Yingguang

2014-08-01

343

Bioreactor Strategy in Bone Tissue Engineering: Pre-Culture and Osteogenic Differentiation Under Two Flow Configurations  

PubMed Central

Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both pre-culture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome.

Kim, Junho

2012-01-01

344

Mesenchymal stem cells from patients to assay bone graft substitutes.  

PubMed

Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg(2+) ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg(2+) and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg(2+) apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase. PMID:23129455

Manfrini, M; Di Bona, C; Canella, A; Lucarelli, E; Pellati, A; D'Agostino, A; Barbanti-Bròdano, G; Tognon, M

2013-06-01

345

Radioresistance of Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines Derived from Nrf2-/- Homozygous Deletion Recombinant-Negative Mice  

PubMed Central

Aim: We determined whether bone marrow from Nrf2?/? compared with Nrf2+/+ mice differed in response to the oxidative stress of continuous marrow culture, and in radiosensitivity of derived stromal and interleukin-3 (IL-3)-dependent hematopoietic progenitor cells. Materials and Methods: Hematopoiesis longevity in Nrf2?/? was compared with Nrf2+/+ mice in long-term bone marrow cultures. Clonogenic irradiation survival curves were performed on derived cell lines. Total antioxidant capacity at baseline in nonirradiated cells and at 24 hours after 5 Gy and 10 Gy irradiation was quantitated using an antioxidant reductive capacity assay. Results: Long-term cultures of bone marrow from Nrf2?/? compared to Nrf2+/+ mice demonstrated equivalent longevity of production of total cells and hematopoietic progenitor cells forming multi-lineage hematopoietic colonies over 26 weeks in culture. Both bone marrow stromal cell lines and Il-3-dependent hematopoietic progenitor cell lines derived from Nrf2?/? mouse marrow cultures were radioresistant compared to Nrf2+/+-derived cell lines. Both DNA repair assay and total antioxidant capacity assay showed no defect in Nrf2?/? compared to Nrf2+/+ stromal cells and IL-3-dependent cells. Conclusion: The absence of a functional Nrf2 gene product does not alter cellular interactions in continuous marrow culture, nor response to dsDNA damage repair and antioxidant response. However, lack of the Nrf2 gene does confer radioresistance on marrow stromal and hematopoietic cells.

BERHANE, HEBIST; EPPERLY, MICHAEL W.; CAO, SHAONAN; GOFF, JULIE P.; FRANICOLA, DARCY; WANG, HONG; GREENBERGER, JOEL S.

2014-01-01

346

Differentiation and Development in Plant Cell Cultures.  

National Technical Information Service (NTIS)

Differentiation in plant cell cultures was studied by tissue culture and genetic approaches. The influence of various environmental factors on differentiation was elucidated using Digitalis lanata protoplasts in the first part of the study. The molecular ...

R. Puupponen-Pimiae

1995-01-01

347

Cross-Talk between CLL Cells and Bone Marrow Endothelial Cells: Role of Signal Transducer and Activator of Transcription-3  

PubMed Central

Summary Chronic lymphocytic leukemia (CLL) bone marrow is characterized by increased angiogenesis. However, the molecular mediators of neovascularization and the biological significance of increased endothelial cell proliferation in CLL require further investigation. Because signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL we studied the role of STAT3 in modulating vascular endothelial growth factor (VEGF) expression and the effect of vascular endothelial cells on CLL cells. Using chromatin immunoprecipitation (ChIP) we found that anti-STAT3 antibodies immunoprecipitated DNA of STAT3, VEGF and other STAT3-regulated genes. In addition, STAT3-short interfering RNA significantly reduced mRNA levels of VEGF in CLL cells suggesting that STAT3 induces VEGF expression in CLL. Remarkably, bone marrow CLL cells expressed high levels of VEGF and high VEGF levels were detected in the plasma of patients with untreated CLL and correlated with white blood cell count. CLL bone marrow biopsies revealed increased microvascular density and attachment of CLL cells to endothelial cells. Co-culture of CLL and human umbilical vein endothelial cells (HUVEC) cells showed a similar attachment. Furthermore, co-culture studies with HUVEC showed that HUVEC protected CLL cells from spontaneous apoptosis by direct cell-to-cell contact as assessed by flow cytometry using Annexin V. Our data suggest that constitutively activated STAT3 induces VEGF production by CLL cells and CLL cells derive a survival advantage from endothelial cells via cell-to cell contact.

Badoux, Xavier; Bueso-Ramos, Carlos; Harris, David; Li, Ping; Liu, Zhiming; Burger, Jan; O'Brien, Susan; Ferrajoli, Alessandra; Keating, Michael J.; Estrov, Zeev

2014-01-01

348

Use of a licensed electrolyte solution as an alternative to tissue culture medium for bone marrow collection.  

PubMed

Bone marrow for transplantation is traditionally collected into tissue culture medium with heparin. A licensed electrolyte solution (Plasma-Lyte A [PLA]) was used as a substitute for tissue culture medium in the harvesting of 28 bone marrows, 17 autologous and 11 allogeneic, which were subsequently transplanted. Data that were analyzed from the 25 evaluable patients consisted of the numbers of cells and colony-forming units in the transplanted marrow as well as the time to neutrophil and platelet engraftment. These results were compared with those in the 30 (26 evaluable) preceding transplanted marrows that were collected into a tissue culture medium (RPMI-1640 [RPMI]). The autologous marrow transplant patients in both the PLA and RPMI groups reached a neutrophil count of > or = 0.5 x 10(9) per L a mean of 19 days following transplantation. The patients who underwent transplantation with allogeneic bone marrow collected in RPMI achieved > or = 0.5 x 10(9) per L of neutrophils an average of 20 days following transplantation, while those who received marrow collected in PLA achieved engraftment of neutrophils to that level in a mean of 21 days. Because in vitro and in vivo results with RPMI and PLA are similar in this study, further work using a licensed solution for clinical bone marrow transplantation is indicated. PMID:8333019

Areman, E M; Dickerson, S A; Kotula, P L; Spitzer, T R; Sacher, R A

1993-07-01

349

Cytokines and growth factors which regulate bone cell function  

NASA Astrophysics Data System (ADS)

Everybody knows that growth factors are most important in making bone. Hormones enhance bone formation from a long distance. Growth factors promote bone formation as an autocrine or paracrine factor in nearby bone. BMP-2 through BMP-8 are in the TGF-? family. BMP makes bone by enchondral ossification. In bone, IGF-II is most abundant, second, TGF-?, and third IGF-I. TGF-? enhances bone formation mainly by intramembranous ossification in vivo. TGF-? affects both cell proliferation and differentiation, however, TGF-? mainly enhances bone formation by intramembranous ossification. Interestingly, TGF-? is increased by estrogen(E 2), androgen, vitamin D, TGF-? and FGF. IGF-I and IGF-II also enhance bone formation. At present it remains unclear why IGF-I is more active in bone formation than IGF-II, although IGF-II is more abundant in bone compared to IGF-I. However, if only type I receptor signal transduction promotes bone formation, the strong activity of IGF-I in bone formation is understandable. GH, PTH and E 2 promotes IGF-I production. Recent data suggest that hormones containing vitamin D or E 2 enhance bone formation through growth factors. Therefore, growth factors are the key to clarifying the mechanism of bone formation.

Seino, Yoshiki

350

Bone Morphogenetic Protein 4 Mediates Human Embryonic Germ Cell Derivation  

PubMed Central

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)–polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

Hiller, Marc; Liu, Cyndi; Blumenthal, Paul D.; Gearhart, John D.

2011-01-01

351

Formation of engineered bone with adipose stromal cells from buccal fat pad.  

PubMed

A robust method for inducing bone formation from adipose-derived stromal cells (ADSCs) has not been established. Moreover, the efficacy of strong osteogenic inducers including BMP-2 for ADSC-mediated bone engineering remains controversial. Meanwhile, the buccal fat pad (BFP), which is found in the oral cavity as an adipose-encapsulated mass, has been shown to have potential as a new accessible source of ADSCs for oral surgeons. However, to date, there have been no reports that define the practical usefulness of ADSCs from BFP (B-ADSCs) for bone engineering. Here, we report an efficient method of generating bone from B-ADSCs using rhBMP-2. The analyses show that B-ADSCs can differentiate in vitro toward the osteoblastic lineage by the addition of rhBMP-2 to culture medium, regardless of the presence of osteoinductive reagents (OSR), as demonstrated by measurements of ALP activity, in vitro calcification, and osteogenic gene expression. Interestingly, adipogenic genes were clearly detectable only in cultures with rhBMP-2 and OSR. However, in vivo bone formation was most substantial when B-ADSCs cultured in this condition were transplanted. Thus, B-ADSCs reliably formed engineered bone when pre-treated with rhBMP-2 for inducing mature osteoblastic differentiation. This study supports the potential translation for B-ADSC use in the clinical treatment of bone defects. PMID:22538411

Shiraishi, T; Sumita, Y; Wakamastu, Y; Nagai, K; Asahina, I

2012-06-01

352

Committed neural progenitor cells derived from genetically modified bone marrow stromal cells ameliorate deficits in a rat model of stroke  

Microsoft Academic Search

Bone marrow stromal cells (MSCs) are an excellent source of cells for treating a variety of central nervous system diseases. In this study, we report the efficient induction of committed neural progenitor cells from rat and human MSCs (NS-MSCs) by introduction of cells with the intracellular domain of Notch-1 followed by growth in the free-floating culture system. NS-MSCs successfully formed

Makoto Hayase; Masaaki Kitada; Shohei Wakao; Yutaka Itokazu; Kazuhiko Nozaki; Nobuo Hashimoto; Yasushi Takagi; Mari Dezawa

2009-01-01

353

Cell Mechanisms of Bone Tissue Loss Under Space Flight Conditions  

Microsoft Academic Search

Investigations on the space biosatellites has shown that the bone skeleton is one of the most im-portant targets of the effect space flight factors on the organism. Bone tissue cells were studied by electron microscopy in biosamples of rats' long bones flown on the board american station \\

Natalia Rodionova

2010-01-01

354

Identification of cultured progenitor cells from human marrow stroma.  

PubMed

The marrow stromal cells (MSC) are essential for regulation of bone remodeling and hematopoiesis. It is of prime importance to isolate MSC and to expand the proliferating cells ex vivo. In this study, we analyzed cultured MSC for various cellular parameters, including cell morphology, cell cycle, and expression of cell surface antigens by flow cytometry. MSC were divided based on cell size to small (S-cells) and large (L-cells) and were visualized by light and electron microscope. The S-cells were proliferating cells correlated with G0/G1 phase of cell cycle, and expressed cFOS. The expression of surface markers CD-34, -44, -51, -61, -62E, -62P, -62L was quantified using flow cytometry. CD-44 was ubiquitously expressed by S and L cells, CD-51 and -61 were expressed by 30%-38% of S-cells. CD-34 and -62 expressed 20% positive of the analyzed cells that were of the proliferating progenitors (S-cells). This study enables the identification of subpopulations from MSC with special attention paid to the proliferating cells from ex vivo cultures of marrow stroma. PMID:12210721

Shur, I; Marom, R; Lokiec, F; Socher, R; Benayahu, D

2002-01-01

355

Insulin-like growth factor I has independent effects on bone matrix formation and cell replication  

SciTech Connect

The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of (2,3-/sup 3/H)proline and (methyl-/sup 3/H)thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on (/sup 3/H)proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on (/sup 3/H)thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis.

Hock, J.M.; Centrella, M.; Canalis, E.

1988-01-01

356

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

Microsoft Academic Search

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

Arshak R. Alexanian

2005-01-01

357

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

PubMed

Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vi