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1

The Effect of Spaceflight on Bone Cell Cultures  

NASA Technical Reports Server (NTRS)

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

Landis, William J.

1999-01-01

2

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

PubMed Central

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

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

2014-01-01

3

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

Microsoft Academic Search

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

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

2009-01-01

4

[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

5

Effect of interleukin 3 on the differentiation and histamine content of cultured bone marrow mast cells  

Microsoft Academic Search

Mouse bone marrow hematopoietic stem cells were isolated from mouse femur bone and cultured in RPMI 1640 supplemented medium with 20 units\\/ml of the purified T-cell lymphokine, interleukin 3 (IL-3). IL-3 was uniquely able to induce the proliferation and differentiation of mature mast cellsin vitro. The sparse granulation of the bone marrow-derived mast cells (BMMC) can be seen by day

H. F. Chiu; B. A. Burrall

1990-01-01

6

Bone grafts engineered from human adipose-derived stem cells in perfusion bioreactor culture.  

PubMed

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 x 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 microm/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-beta-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture, and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs. PMID:19678762

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

2010-01-01

7

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. PMID:24369019

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

2013-01-01

8

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

9

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

PubMed

Osteoprogenitor cells combined with supportive biomaterials represent a promising approach to advance the standard of care for bone grafting procedures. However, this approach faces challenges, including inconsistent bone formation, cell survival in the implant, and appropriate biomaterial degradation. We have developed a collagen-hydroxyapatite (HA) scaffold that supports consistent osteogenesis by donor-derived osteoprogenitors, and is more easily degraded than a pure ceramic scaffold. Herein, the material properties are characterized as well as cell attachment, viability, and progenitor distribution in vitro. Furthermore, we examined the biological performance in vivo in a critical-size mouse calvarial defect. To aid in the evaluation of the in-house collagen-HA scaffold, the in vivo performance was compared with a commercial collagen-HA scaffold (Healos(®) , Depuy). The in-house collagen-HA scaffold supported consistent bone formation by predominantly donor-derived osteoblasts, nearly completely filling a 3.5 mm calvarial defect with bone in all samples (n?=?5) after 3 weeks of implantation. In terms of bone formation and donor cell retention at 3 weeks postimplantation, no statistical difference was found between the in-house and commercial scaffold following quantitative histomorphometry. The collagen-HA scaffold presented here is an open and well-defined platform that supports robust bone formation and should facilitate the further development of collagen-hydroxyapatite biomaterials for bone tissue engineering. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 243-253, 2015. PMID:24909953

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

2015-02-01

10

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

11

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

12

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

PubMed

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

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

2014-01-01

13

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

PubMed Central

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

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

2014-01-01

14

Ki-Energy (Life-Energy) Stimulates Osteoblastic Cells and Inhibits the Formation of Osteoclast-Like Cells in Bone Cell Culture Models  

Microsoft Academic Search

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese

S. Tsuyoshi Ohnishi; Kozo Nishino; Satoshi Uchiyama; Tomoko Ohnishi; Masayoshi Yamaguchi

2007-01-01

15

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

16

[Comparative characterization of mesenchymal bone marrow stromal cells at early and late stages of culturing].  

PubMed

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1-4) but disappear at later stages (passages 9-11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6). PMID:18946989

Kozhevnikova, M N; Mikaelian, A S; Paiushina, O V; Starostin, V I

2008-01-01

17

Mechanical stretching increases the number of cultured bone cells synthesizing DNA and alters their pattern of protein synthesis  

Microsoft Academic Search

Summary  A simple method was devised for applying mechanical stretching to bone cell cultures. Bone cells cultured on the flexible\\u000a plastic membrane of a Petriperm dish are placed over a template with a convex surface. A lead weight is then placed on top\\u000a of the dish which causes the membrane and the tightly attached cells to be stretched. Mechanical stretching, applied

Shin Hasegawa; S. Sato; S. Saito; Y. Suzuki; D. M. Brunette

1985-01-01

18

Human triple cell co-culture for evaluation of bone implant materials.  

PubMed

Central to the formation of tissue at implant surfaces are the interactions between multiple cell types including fibroblasts, endothelial cells and, in the case of bone, cells of the osteoblastic lineage. To date the importance of population dynamics and interactions have been largely neglected in the in vitro evaluation of biomaterials. To fill this gap we have developed a co-culture system using 3 cell types, primary human bone marrow stromal cells (HBMC), microvascular endothelial cells (HMVEC) and abdominal dermal fibroblasts (HDF). Proliferation of each cell type separately and differentiation of HBMC were determined by flow cytometry analysis. The medium used promoted HBMC differentiation toward osteoblasts without affecting the state of differentiation of HDF and HMVEC. Furthermore, HBMC are strongly affected by HDF and HMVEC, and vice versa. When used on a titanium coated substrate the triple cell culture system identified preferential HBMC proliferation relative to HDF if HMVEC was present. This developed culture system represents a new, optimised and potentially predictive approach to evaluate biomaterial biocompatibility early in development. PMID:23420382

Wein, Frederik; Bruinink, Arie

2013-04-01

19

Enhanced Differentiation of Adult Bone Marrow-Derived Stem Cells to Liver Lineage in Aggregate Culture  

PubMed Central

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.

2011-01-01

20

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

21

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

22

Effects of Initial Cell Density and Hydrodynamic Culture on Osteogenic Activity of Tissue-Engineered Bone Grafts  

PubMed Central

This study aimed to study the effects of initial cell density and in vitro culture method on the construction of tissue-engineered bone grafts and osteogenic activities. Human mesenchymal stem cells (hMSCs) were seeded onto cubic scaffolds prepared from demineralized bone matrix (DBM) by three methods - static, hydrodynamic, or fibrin hydrogel-assisted seeding. The resulting cell-scaffold constructs were cultured in vitro by static flask culture or hydrodynamic culture. The initial cell density and the subsequent in vitro proliferation and alkaline phosphate activities of the constructs were analyzed. The constructs were also subcutaneously implanted in nude mice to examine their in vivo osteogenic activities. Hydrogel-assisted seeding gave the highest seeding efficiency, followed by hydrodynamic and conventional static seeding. During in vitro culture, hydrodynamic culture produced higher plateau cell densities, alkaline phosphatase (ALP) activities, and extracellular matrix production than static culture. After subcutaneous implantation in nude mice, the implants prepared by the combination of hydrogel-assisted seeding and hydrodynamic culture produced higher wet weight and bone mineral density than implants prepared by other methods. The results suggest that the hydrogel-assisted seeding can substantially increase the initial seed cell density in scaffolds. Subsequent hydrodynamic culture can promote the proliferation and osteoblastic differentiation of the seeded cells. Correspondingly, bone grafts produced by the combination of these two methods achieved the highest osteogenic activity among the three methods employed. PMID:23326488

Luo, Fei; Hou, Tian-Yong; Zhang, Ze-Hua; Xie, Zhao; Wu, Xue-Hui; Xu, Jian-Zhong

2013-01-01

23

Isolation, culture, and induced multiple differentiation of Mongolian sheep bone marrow-derived mesenchymal stem cells.  

PubMed

The aim of this paper was to explore the optimal method of isolating, purifying, and proliferating Mongolian sheep bone marrow-derived mesenchymal stem cells (BMSCs) and their multiple differentiation potentialities. Bone marrow (BM) was punctured from ?1-year-old sheep, and BMSCs were harvested through gradient centrifuge and adherent cultures. Analysis of the growth of the passage 1, 5, and 10 cultures revealed an S-shaped growth curve with a population doubling time of 31.2 h. Karyotyping indicated that the chromosome number in the Mongolian sheep was 2n?=?54, comprising 26 pairs of autosomes and one pair of sex chromosomes (XY). RT-PCR demonstrated that OCT4, SOX2, and Nanog genes at passage 3 were positively expressed. The P3 BMSCs were cultured in vitro under inductive environments and induced into adipocytes, osteoblasts, chondrocytes, neural cells, and cardiomyocytes. Their differentiation properties were confirmed by histological staining, such as oil red, Alizarin red, hematoxylin-eosin, toluidine blue, and periodic acid schiff. RT-PCR showed that the specific genes to be induced were all expressed. This proves that the isolated cells are indeed the BMSCs and also provides valuable materials for somatic cell cloning and transgenic research. PMID:24399254

Liu, Zongzheng; Wang, Wei; Gao, Jinfang; Zhou, Huanmin; Zhang, Yanru

2014-05-01

24

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

SciTech Connect

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

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

1987-06-01

25

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

26

Cell Stem Cell Endogenous Bone Marrow MSCs  

E-print Network

). The existence of multipotent bone marrow stromal cells (BMSCs), or skeletal/mesenchymal stem cells (SSCs of cultured cells (Sacchetti et al., 2007). Similar multipotent MSCs can be isolated from mouse bone marrowCell Stem Cell Article Endogenous Bone Marrow MSCs Are Dynamic, Fate-Restricted Participants

Mootha, Vamsi K.

27

Effect of osteoclast co-culture on the differentiation of human mesenchymal stem cells grown on bone graft granules.  

PubMed

Traditional approaches to bone repair are currently being integrated with innovative tissue-engineering techniques, as researchers and clinicians shift their treatment focus toward regenerating functional tissue rather than just filling a defect to provide structural support. Cells are expanded and incorporated into implantable systems in hopes of enhancing the bone-forming capabilities of traditional bone graft substitutes. The present study examined how osteoclasts might be used to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into bone forming cells. The two cell types were co-cultured on a resorbable, three-dimensional bone graft substitute. Osteoclasts were seeded prior to the addition of hMSCs, as well as simultaneously, to determine if resorption of the scaffold would have any bearing on observed response by hMSCs. When seeded directly with hMSCs on the 3-D substrates, the osteoclasts had an increase in TRAP expression over time if seeded simultaneously. The co-culture setup had a positive influence on the proliferation of hMSCs. Late stage osteoblast differentiation markers (bone sialoprotein) were positively affected by direct co-culture with osteoclasts. The addition of RANKL to the culture medium for osteoclastogenesis appears to be a factor in the observed responses by hMSCS, but is not the only factor influencing the MSCs. Osteoclasts were shown to have an influence on the development of mesenchymal stem cells into osteoblasts when cultured in vitro. Findings from this study, coupled with the knowledge obtained from our previous work, will aid in the development of a clinically viable mesenchymal stem cell based bone graft system. PMID:20566059

Sinclair, Sarina S Kay; Burg, Karen J L

2011-01-01

28

Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells  

Microsoft Academic Search

Bone formation around implants is influenced by surface geometry. Since cell\\/matrix\\/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and

Paulo Tambasco de Oliveira; Antonio Nanci

2004-01-01

29

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system  

Microsoft Academic Search

ObjectivePluripotent mesenchymal stem cells (MSC) have been isolated and well characterized from several tissue sources, including bone marrow stroma. MSC from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated MSC from bovine bone marrow and induced chondrogenesis in order to establish a new experimental model of stem cell research.

Darko Bosnakovski; Morimichi Mizuno; Gonhyung Kim; Taketo Ishiguro; Masahiro Okumura; Toshihiko Iwanaga; Tsuyoshi Kadosawa; Toru Fujinaga

2004-01-01

30

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

31

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

SciTech Connect

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

Perkins, S.; Fleischman, R.A.

1988-04-01

32

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

PubMed

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

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

2013-12-01

33

Isolation, culture, and osteogenic/chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.  

PubMed

Musculoskeletal disorders, as non-healing fractures and large bone defects, articular cartilage and subchondral bone injuries, often result in lifelong chronic pain and compromised quality of life. Although generally a natural process, failure of large bone defects to heal such as after complex fractures, resection of tumours, infections, or revisions of joint replacements remains a critical challenge that requires more appropriate solutions as those currently available. In addition, regeneration of chondral and osteochondral defects continues to be a challenge until to date. A profound understanding of the underlying mechanisms of endogenous regeneration is a prerequisite for successful bone and cartilage regeneration. Presently, one of the most promising therapeutic approaches is cell-based tissue engineering which provides a healthy population of cells to the injured site. Use of differentiated cells has severe limitations; an excellent alternative would be the application of adult marrow stromal cells/mesenchymal stem cells (MSC) which possess extensive proliferation potential and proven capability to differentiate along the osteochondral pathway. The process of osteo-/chondrogenesis can be mimicked in vitro by inducing osteo-chondroprogenitor stem cells to undergo osteogenesis and chondrogenesis through exposure of osteo-/chondrogenic favourable microenvironmental, mechanical, and nutritional conditions. This chapter provides comprehensive protocols for the isolation, expansion, and osteo-/chondrogenic differentiation of adult bone marrow-derived MSC. PMID:22610563

Grässel, Susanne; Stöckl, Sabine; Jenei-Lanzl, Zsuzsa

2012-01-01

34

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

SciTech Connect

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

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

2010-04-02

35

Prevention of Liver Fibrosis by Intrasplenic Injection of High-Density Cultured Bone Marrow Cells in a Rat Chronic Liver Injury Model  

PubMed Central

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

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

2014-01-01

36

Proteomic analysis of the impact of static culturing on the expansion of rat bone marrow mesenchymal stem cells.  

PubMed

The clinical potential of mesenchymal stem cells (MSC) in tissue engineering and regenerative medicine is due to their self-renewal, proliferation and multi-lineage differentiation potential. Clinical use requires large cell numbers; which can, theoretically, be generated by ex vivo expansion of plastic adherent, MSC subpopulation, of bone marrow cells (BMC). Effects of serial culture on MSC phenotype were investigated using non-gel based quantitative proteomic methodology for static monolayer cultures of rat BMC. In total, 382 proteins were relatively quantified (? 2 peptides). Nine proteins were up-regulated and seven down-regulated at passage 4 relative to passage 2 (p ? 0.05). We propose that serial culture impacts on MSC expansion (observed decline in colony forming potential and colony size) is through a combination of osteogenic differentiation and ageing/senescence and propose six novel protein biomarkers as candidates for quality control purposes in bioprocessing. PMID:22566207

Peter, Stefan; Evans, Caroline; Ow, Saw Y; Scutt, Andy M; Wright, Phillip C; Biggs, Catherine A

2012-08-01

37

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

SciTech Connect

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

Aguirre, A.; Planell, J.A. [Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15-21, 08028 Barcelona (Spain) [Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15-21, 08028 Barcelona (Spain); Dept. of Material Science and Metallurgical Engineering, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); CIBER-BBN, Maria de Luna 11, Ed. CEEI, 50118 Zaragoza (Spain); Engel, E., E-mail: elisabeth.engel@upc.edu [Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15-21, 08028 Barcelona (Spain); Dept. of Material Science and Metallurgical Engineering, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); CIBER-BBN, Maria de Luna 11, Ed. CEEI, 50118 Zaragoza (Spain)

2010-09-17

38

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. PMID:20091907

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

2010-01-01

39

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

40

Stimulation of creatine kinase BB activity by parathyroid hormone and by prostaglandin E2 in cultured bone cells.  

PubMed Central

Bone cells in culture responded to parathyroid hormone (PTH) and prostaglandin E2 (PGE2) by a 2-fold increase in creatine kinase (CK) activity. Combined treatment resulted in a higher response than with PTH alone. Calcitonin (CT) failed to stimulate CK activity, did not affect the response of CK to PTH, but inhibited slightly the increase in CK activity by PGE2. Bone-cell cultures grown in low [Ca2+] (0.125 mM), enriched in PTH-responsive osteoblast-like cells, responded to PTH, but not to PGE2 or CT, by increased CK activity. In both normal and low-[Ca2+] cultures, 8-bromo cyclic AMP did not affect CK activity, nor did it change the response of the cells to PTH, PGE2 or CT. The increase in CK activity was time- and dose-dependent and inhibited both by cycloheximide and by actinomycin D. The isoenzyme of CK stimulated was the CKBB form, the isoenzyme induced by other hormones. This appears to be the first report of the stimulation of CK activity by a polypeptide hormone or a prostaglandin. We suggest that stimulation of CKBB can serve as a marker for the action of a variety of hormones and growth promoters. PMID:3856427

Sömjen, D; Kaye, A M; Binderman, I

1985-01-01

41

Human Bone Marrow and Umbilical Cord Blood Cells Generate CD4^+ and CD8^+ Single-Positive T Cells in Murine Fetal Thymus Organ Culture  

Microsoft Academic Search

Murine fetal thymus lobes isolated from both normal and scid\\/scid mice can be colonized by donor cells from either human bone marrow or human umbilical cord blood in vitro. Subsequent organ culture results in a transient production of a few CD4^+ CD8^+ (double-positive) cells and then the accumulation of CD4^+ or CD8^+ (single-positive) T cells. A significant number of immature

Helen Yeoman; Ronald E. Gress; Catherine V. Bare; Anne G. Leary; Edward A. Boyse; Judith Bard; Leonard D. Shultz; David T. Harris; Dominick Deluca

1993-01-01

42

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.

43

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. 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

44

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

45

Establishment and validation of an in vitro co-culture model to study the interactions between bone and prostate cancer cells  

Microsoft Academic Search

Bone is the preferred site for prostate cancer (PCa) metastases. Once the tumor has established itself within the bone there\\u000a is virtually no cure. To better understand the interactions between the PCa cells and bone environment in the metastatic process\\u000a new model systems are needed. We have established a two-compartment in vitro co-culturing model that can be used to follow

Annika Nordstrand; Jonas Nilsson; Åse Tieva; Pernilla Wikström; Ulf H. Lerner; Anders Widmark

2009-01-01

46

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

47

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

PubMed

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

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

2014-09-01

48

Cultures of human osteoblastic cells from dialysis patients: influence of bone turnover rate on in vitro selection of interleukin-6 and osteoblastic cell makers.  

PubMed

The factors contributing to renal osteodystrophy are still incompletely characterized. A variety of cytokines and growth factors appear to have ill-defined roles in this disease. Our aim is to compare osteoblastic cell growth and different osteoblastic markers in vitro with histomorphometric bone parameters and some serum bone-turnover markers in vivo in dialysis patients with either high- (HTBD) or low-turnover (LTBD) bone disease. Six patients were diagnosed to have LTBD, and another five patients, HTBD. Intact parathyroid hormone (PTH) and osteocalcin (OC) levels in serum were greater in patients with HTBD than in those with LTBD. Osteoblastic cells isolated from iliac crest biopsy specimens were grown in culture medium for different times up to 13 days. Osteoblastic cell growth (cell number and area under the cell growth curve) was greater in patients with HTBD than in those with LTBD. Static and dynamic bone formation parameters correlated with serum PTH levels. No correlation was found between PTH and osteoblastic cell proliferation. OC, C-terminal type I procollagen, and alkaline phosphatase osteoblastic secretion in vitro were similar in the HTBD and LTBD groups. However, interleukin-6 (IL-6) secretion was greater in cells isolated from patients with LTBD. Our results indicate that osteoblastic cell growth and osteoblastic IL-6 secretion are related to bone turnover in patients with osteodystrophy. Our findings support the hypothesis that factors other than PTH level might have an important role in affecting osteoblastic function in renal osteodystrophy. PMID:11136164

Sánchez, M C; Bajo, M A; Selgas, R; Mate, A; Sánchez-Cabezudo, M J; López-Barea, F; Esbrit, P; Martínez, M E

2001-01-01

49

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

PubMed

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

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

2013-09-01

50

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

Microsoft Academic Search

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

SUN-KYEONG LEE; JOSEPH A. LORENZO

1999-01-01

51

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

PubMed

In this study, rat bone marrow cells (RBM) were used to evaluate different titanium and hydroxyapatite dental implant surfaces. The implant surfaces investigated were: a titanium surface having a porous titanium plasma-sprayed coating (sample code Ti-TPS), a titanium surface with a deep profile structure (sample code Ti-DPS), an uncoated titanium substrate with a machined surface (sample code Ti-ma) and a machined titanium substrate with a porous hydroxyapatite plasma-sprayed coating (sample code Ti-HA). RBM cells were cultured on the disc-shaped test substrates for 14 days. The culture medium was changed daily and examined for calcium and phosphate concentrations. After 14 days specimens were examined by light microscopy, scanning electron microscopy, energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. All test substrates facilitated RBM growth of extracellular matrix formation. Ti-DPS and Ti-TPS to the highest degree, followed by Ti-ma and Ti-HA. Ti-DPS and Ti-TPS displayed the highest cell density and thus seem to be well suited for the endosseous portion of dental implants. RBM cells cultured on Ti-HA showed a delayed growth pattern. This may be related to its high phosphate ion release. PMID:12102195

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

2002-08-01

52

Effect of vasopressin, oxytocin and LHRH on the proliferation and metabolism of rat bone marrow stromal cells in culture.  

PubMed

Effect of vasopressin, oxytocin and LHRH (10 and 20 pg/ml medium) on the proliferation and metabolism of cultured rat bone marrow stromal cells was investigated by methyl-3H-thymidine incorporation, cytochemistry and estimation of enzyme activities. Vasopressin did not change of the activity of tetrahydrofolate dehydrogenase (4HFDH), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD) and the level of reduced glutathione (GSH). However, the higher concentration of vasopressin significantly lowered the activity of acetylcholinesterase (AchE). As compared with the control cultures, stromal cells grown in the presence of oxytocin showed higher (at lower hormone concentration) and lower (at higher concentration) LDH activity as well as lower G6PD activity (only at higher concentration), while the activity of AchE and the level of GSH was not changed. LHRH significantly increased G6PD and AchE activity and decreased LDH activity in the cultured cells. As revealed by cytochemistry, LHRH specifically enhanced 4HFDH activity in reticular cells. PMID:1764608

Miszta, H; Kasprzykowski, F; Grzonka, Z; Lankiewicz, L

1991-09-01

53

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. PMID:52158

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

1975-01-01

54

Bone Sialoprotein (BSP) is a Crucial Factor for the Expression of Osteoblastic Phenotypes of Bone Marrow Cells Cultured on Type I Collagen Matrix  

Microsoft Academic Search

.   In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow\\u000a cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other\\u000a hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were\\u000a harvested from rat femur and

M. Mizuno; T. Imai; R. Fujisawa; H. Tani; Y. Kuboki

2000-01-01

55

Flow and nutrient transport through porous scaffolds used for the culture of bone cells in perfusion bioreactors  

NASA Astrophysics Data System (ADS)

The goal is to understand via computation the behavior of the flow inside porous scaffolds that are used in bone tissue bioreactors. Fluid shear is an important stimulatory factor in preosteoblastic cells seeded in scaffolds and cultured under continuous flow perfusion. A Lattice Boltzmann method has been employed to simulate the flow field within porous scaffolds obtained with high resolution micro-CT. Lagrangian methods have also been used to determine the nutrient dispersion inside the scaffolds. The shear stresses calculated inside the scaffold architecture indicate that the shear stresses experienced by cells inside the scaffold can vary by orders of magnitude. This is important when designing scaffolds for bone tissue growth, since osteoblastic cells require to be stimulated by shear for growth. Moreover, cell detachment can occur when the fluid shear is too high, thus, placing a limit on the stresses that a particular scaffold design should allows. The talk will address the methodology, the validation and the correlation of scaffold structure characteristics with the shear stresses and with the rate of mass transfer.

Papavassiliou, Dimitrios; Voronov, Roman; Sikavitsas, Vassilios; Vangordon, Samuel

2009-03-01

56

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. PMID:23730368

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

2013-01-01

57

Construction of Mesenchymal Stem Cell–Containing Collagen Gel with a Macrochanneled Polycaprolactone Scaffold and the Flow Perfusion Culturing for Bone Tissue Engineering  

PubMed Central

Abstract A novel bone tissue-engineering construct was developed by using poly(?-caprolactone) (PCL)-macrochanneled scaffolds combined with stem cell-seeded collagen hydrogels and then applying flow perfusion culture. Rat mesenchymal stem cells (MSCs) were loaded into collagen hydrogels, which were then combined with macrochanneled PCL scaffolds. Collagen hydrogels were demonstrated to provide favorable growth environments for MSCs and to foster proliferation. Cell number determination identified retention of substantially fewer (50–60%) cells when they were seeded directly onto macrochanneled PCL than of cells engineered within collagen hydrogels. Additionally, the cells actively proliferated within the combined scaffold for up to 7 days. MSC-loaded collagen–PCL scaffolds were subsequently cultured under flow perfusion to promote proliferation and osteogenic differentiation. Cells proliferated to levels significantly higher in flow perfusion culture than that under static conditions during 21 days. A quantitative polymerase chain reaction (QPCR) assay revealed significant alterations in the transcription of bone-related genes such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP), such as 8-, 2.5-, and 3-fold induction, respectively, after 10 days of flow perfusion relative to those in static culture. OPN and OCN protein levels, as determined by Western blot, increased under flow perfusion. Cellular mineralization was significantly enhanced by the flow perfusion during 21 and 28 days. Analyses of mechanosensitive gene expression induced by flow perfusion shear stress revealed significant upregulation of c-fos and cyclooxygenase-2 (COX-2) during the initial culture period (3–5 days), suggesting that osteogenic stimulation was possible as a result of mechanical force-driven transduction. These results provide valuable information for the design of a new bone tissue-engineering system by combining stem cell-loaded collagen hydrogels with macrochanneled scaffolds in flow perfusion culture. PMID:23515189

Yu, Hye-Sun; Won, Jong-Eun; Jin, Guang-Zhen

2012-01-01

58

Pro-elastogenic effects of bone marrow mesenchymal stem cell-derived smooth muscle cells on cultured aneurysmal smooth muscle cells.  

PubMed

Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non-surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and -9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM-MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM-MSC-derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM-MSC-derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow-up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long-term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25376929

Swaminathan, Ganesh; Gadepalli, Venkat S; Stoilov, Ivan; Mecham, Robert P; Rao, Raj R; Ramamurthi, Anand

2014-11-01

59

Culture medium of bone marrow-derived human mesenchymal stem cells effects lymphatic endothelial cells and tumor lymph vessel formation  

PubMed Central

Human bone marrow mesenchymal stem cells (hBM-MSCs) favor tumor growth and metastasis in vivo and in vitro. Neovascularization is involved in several pathological conditions, including tumor growth and metastasis. Previous studies have demonstrated that human bone marrow MSC-derived conditioned medium (hBM-MSC-CM) can promote tumor growth by inducing the expression of vascular epidermal growth factor (VEGF) in tumor cells. However, the effect of BM-MSCs on tumor lymph vessel formation has yet to be elucidated. In the present study, the effect of BM-MSCs on processes involved in lymph vessel formation, including tube formation, migration and proliferation, was investigated in human-derived lymphatic endothelial cells (HDLECs). It was identified that hBM-MSC-CM promoted the tube formation and migration of HDLECs. In addition, tumor cells were revealed to participate in lymph vessel formation. In the present study, the SGC-7901, HGC-27 and GFP-MCF-7 cell lines were treated with hBM-MSC-CM. The results demonstrated that the expression of the lymph-associated markers, prospero homeobox protein 1 and VEGF receptor-3, were increased in the SGC-7901 and HGC-27 cell lines, but not in the GFP-MCF-7 cells. The tube formation assay demonstrated that the HGC-27 cells treated with hBM-MSC-CM for 20 days underwent tube formation. These findings indicate that hBM-MSC-CM can promote tube formation in HDLECs and HGC-27 cells, which may be associated with lymph vessel formation during tumor growth and metastasis. PMID:25663886

ZHAN, JIE; LI, YAHONG; YU, JING; ZHAO, YUANYAUN; CAO, WENMING; MA, JIE; SUN, XIAOXIAN; SUN, LI; QIAN, HUI; ZHU, WEI; XU, WENRONG

2015-01-01

60

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

61

An exploratory clinical trial for idiopathic osteonecrosis of femoral head by cultured autologous multipotent mesenchymal stromal cells augmented with vascularized bone grafts.  

PubMed

Idiopathic osteonecrosis of femoral head (ION) is a painful disorder that progresses to collapse of the femoral head and destruction of the hip joint. Although its precise pathology remains unknown, the loss of blood supply causing the loss of living bone-forming cells is a hallmark of the pathophysiology of osteonecrosis. Transplantation of multipotent mesenchymal stromal cells (MSCs) is a promising tool for regenerating the musculoskeletal system. The aim of the present study was to assess the safety and efficacy of transplantation of cultured autologous bone marrow-derived MSCs mixed with ?-tricalcium phosphate (?-TCP) in combination with vascularized bone grafts for the treatment of advanced stage ION in a clinical trial. Ten patients with stage 3 ION were enrolled in this study. Autologous bone marrow-derived MSCs were cultured with autologous serum, and cells (0.5-1.0×10(8)) were transplanted after mixing with ?-TCP granules in combination with vascularized iliac bone grafts. Patients were assessed 24 months after treatment. The primary and secondary endpoints were progression of the radiological stage and changes in bone volume at the femoral head, and clinical score, respectively. Nine of ten patients completed the protocol, seven of whom remained at stage 3, and the remaining two cases progressed to stage 4. The average bone volume increased from 56.5±8.5?cm(3) to 57.7±10.6?cm(3). The average clinical score according to the Japan Orthopaedic Association improved from 65.6±25.5 points to 87.9±19.0 points. One severe adverse event was observed, which was not related to the clinical trial. Although the efficacy of cell transplantation was still to be determined, all procedures were successfully performed and some young patients with extensive necrotic lesions with pain demonstrated good bone regeneration with amelioration of symptoms. Further improvements in our method using MSCs and the proper selection of patients will open a new approach for the treatment of this refractory disease. PMID:24593258

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

2014-08-01

62

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

PubMed Central

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

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

2014-01-01

63

Differentiation-Inducing Factor Purified from Conditioned Medium of Mitogen-Treated Spleen Cell Cultures Stimulates Bone Resorption  

Microsoft Academic Search

Spleen cells treated with mitogens produce a potent bone-resorbing factor called osteoclast-activating factor (OAF). To examine the relationship between the bone-resorbing factor and other protein factors produced by spleen cells, the colony-stimulating factor (CSF), the differentiation-inducing factor (DIF), the macrophage fusion factor (MFF), and the macrophage growth factor (MGF) were purified from 2.68 liters of conditioned medium of mouse spleen

Etsuko Abe; Hirofumi Tanaka; Yoshiko Ishimi; Chisato Miyaura; Takamune Hayashi; Hiroshi Nagasawa; Mikio Tomida; Yuri Yamaguchi; Motoo Hozumi; Tatsuo Suda

1986-01-01

64

Human Bone Marrow Mesenchymal Stem Cells Support the Derivation and Propagation of Human Induced Pluripotent Stem Cells in Culture  

PubMed Central

Abstract 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

2013-01-01

65

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

66

Induction of bone by epithelial cell products  

Microsoft Academic Search

SUMMARY The bones of the head and face of vertebrate embryos only form after their progenitor cells have undergone an inductive interaction with embryonic epithelia. We have investigated whether epithelial cell products can substitute for epithelia in allowing mandibular ecto- mesenchyme to form bone. Mandibular epithelia from embryonic chicks were cultured on Millipore filters for 28 days to allow them

BRTAN K. HALL; R. J. VAN EXAN

67

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. PMID:22684242

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

68

Onset of heterogeneity in culture-expanded bone marrow stromal cells  

E-print Network

Inconsistencies among in vitro and in vivo experiments using adult mesenchymal stem cells (MSCs) confound development of therapeutic, regenerative medicine applications, and in vitro expansion is typically required to ...

Whitfield, Matthew J.

69

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

70

A Trabecular Bone Explant Model of Osteocyte–Osteoblast Co-Culture for Bone Mechanobiology  

PubMed Central

The osteocyte network is recognized as the major mechanical sensor in the bone remodeling process, and osteocyte–osteoblast communication acts as an important mediator in the coordination of bone formation and turnover. In this study, we developed a novel 3D trabecular bone explant co-culture model that allows live osteocytes situated in their native extracellular matrix environment to be interconnected with seeded osteoblasts on the bone surface. Using a low-level medium perfusion system, the viability of in situ osteocytes in bone explants was maintained for up to 4 weeks, and functional gap junction intercellular communication (GJIC) was successfully established between osteocytes and seeded primary osteoblasts. Using this novel co-culture model, the effects of dynamic deformational loading, GJIC, and prostaglandin E2 (PGE2) release on functional bone adaptation were further investigated. The results showed that dynamical deformational loading can significantly increase the PGE2 release by bone cells, bone formation, and the apparent elastic modulus of bone explants. However, the inhibition of gap junctions or the PGE2 pathway dramatically attenuated the effects of mechanical loading. This 3D trabecular bone explant co-culture model has great potential to fill in the critical gap in knowledge regarding the role of osteocytes as a mechano-sensor and how osteocytes transmit signals to regulate osteoblasts function and skeletal integrity as reflected in its mechanical properties. PMID:20827376

Chan, Meilin Ete; Lu, Xin L.; Huo, Bo; Baik, Andrew D.; Chiang, Victor; Guldberg, Robert E.; Lu, Helen H.; Guo, X. Edward

2010-01-01

71

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

72

Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds.  

PubMed

The nonhematopoietic component of bone marrow includes multipotent mesenchymal stem cells (MSC) capable of differentiating into fat, bone, muscle, cartilage, and endothelium. In this report, we describe the cell culture and characterization, delivery system, and successful use of topically applied autologous MSC to accelerate the healing of human and experimental murine wounds. A single bone marrow aspirate of 35-50 mL was obtained from patients with acute wounds (n = 5) from skin cancer surgery and from patients with chronic, long-standing, nonhealing lower extremity wounds (n = 8). Cells were grown in vitro under conditions favoring the propagation of MSC, and flow cytometry and immunostaining showed a profile (CD29+, CD44+, CD105+, CD166+, CD34-, CD45-) highly consistent with published reports of human MSC. Functional induction studies confirmed that the MSC could differentiate into bone, cartilage, and adipose tissue. The cultured autologous MSC were applied up to four times to the wounds using a fibrin polymer spray system with a double-barreled syringe. Both fibrinogen (containing the MSC) and thrombin were diluted to optimally deliver a polymerized gel that immediately adhered to the wound, without run-off, and yet allowing the MSC to remain viable and migrate from the gel. Sequential adjacent sections from biopsy specimens of the wound bed after MSC application showed elongated spindle cells, similar to their in vitro counterparts, which immunostained for MSC markers. Generation of new elastic fibers was evident by both special stains and antibodies to human elastin. The application of cultured cells was safe, without treatment-related adverse events. A strong direct correlation was found between the number of cells applied (greater than 1 x 10(6) cells per cm2 of wound area) and the subsequent decrease in chronic wound size (p = 0.0058). Topical application of autologous MSC also stimulated closure of full-thickness wounds in diabetic mice (db/db). Tracking of green fluorescent protein (GFP)+ MSC in mouse wounds showed GFP+ blood vessels, suggesting that the applied cells may persist as well as act to stimulate the wound repair process. These findings indicate that autologous bone marrow-derived MSC can be safely and effectively delivered to wounds using a fibrin spray system. PMID:17518741

Falanga, Vincent; Iwamoto, Satori; Chartier, Molly; Yufit, Tatyana; Butmarc, Janet; Kouttab, Nicholas; Shrayer, David; Carson, Polly

2007-06-01

73

Mesenchymal derivatives of genetically unstable human embryonic stem cells are maintained unstable but undergo senescence in culture as do bone marrow-derived mesenchymal stem cells.  

PubMed

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here, we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages, respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations, suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs, which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells. PMID:24380659

Karagiannidou, Angeliki; Varela, Ioanna; Giannikou, Krinio; Tzetis, Maria; Spyropoulos, Antonia; Paterakis, George; Petrakou, Eftichia; Theodosaki, Maria; Goussetis, Evgenios; Kanavakis, Emmanuel

2014-02-01

74

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

PubMed Central

AIM: To investigate the interaction between mesenchymal stem cells (MSCs) and bone grafts using two different cultivation methods: static and dynamic. METHODS: MSCs were isolated from rat bone marrow. MSC culture was analyzed according to the morphology, cell differentiation potential, and surface molecular markers. Before cell culture, freeze-dried bone (FDB) was maintained in culture for 3 d in order to verify culture medium pH. MSCs were co-cultured with FDB using two different cultivation methods: static co-culture (two-dimensional) and dynamic co-culture (three-dimensional). After 24 h of cultivation by dynamic or static methods, histological analysis of Cell adhesion on FDB was performed. Cell viability was assessed by the Trypan Blue exclusion method on days 0, 3 and 6 after dynamic or static culture. Adherent cells were detached from FDB surface, stained with Trypan Blue, and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture. Statistical analyses were performed with SPSS and a P < 0.05 was considered significant. RESULTS: The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures. Rat MSCs were positive for CD44, CD90 and CD29 and negative for CD34, CD45 and CD11bc. FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH (P > 0.05). In histological analysis, there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods (P < 0.05). The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method. On day 0, the cell viability in the dynamic system was significantly higher than in the static system (P < 0.05). There was a statistical difference in cell viability between days 0, 3 and 6 after dynamic culture (P < 0.05). In static culture, cell viability on day 6 was significantly lower than on day 3 and 0 (P < 0.05). CONCLUSION: An alternative cultivation method was developed to improve the MSCs adhesion on FDB, demonstrating that dynamic co-culture provides a superior environment over static conditions. PMID:22468180

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

2012-01-01

75

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

Microsoft Academic Search

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

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

2002-01-01

76

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

77

Natural killer (NK) cell activity in human long-term bone marrow cultures (LTBMC): effects of interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the progenitor cells.  

PubMed Central

Human bone marrow-derived progenitor cells were studied in a long-term bone marrow culture system (LTBMC) dependent on an autologous stroma cell layer. The establishment of the stromal cell layer was facilitated by using marrow obtained from small pieces of sternum, which was cultured for 4 weeks without addition of exogenous growth factors. After this period, the response of LTBMC to two different cytokines [recombinant human interleukin-2 (rhIL-2) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF)] was investigated. Our results show proliferation in response to both cytokines and induction of differentiation of cells able to bind IL-2 and/or GM-CSF again. The two cytokines also generate cells responding to rhGM-CSF by colony formation. However, a difference with respect to morphology, phenotype and cytotoxic function of cells in the LTBMC, was noted between the two cytokines. Cells with large granular lymphocyte (LGL) morphology and cytotoxic activity against K562 and Daudi were generated only in the rhIL-2-supplemented LTBMC. This was compatible with a higher frequency of cells expressing the CD56+ phenotype in the IL-2-stimulated LTBMC as compared to the GM-CSF supplemented LTBMC. Our results also demonstrate the existence of a population of myeloid progenitor cells (CD33+) with ability to bind IL-2 in fresh bone marrow (BM). Images Figure 1 Figure 5 PMID:1634251

Sitnicka, E; Hansson, M

1992-01-01

78

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

PubMed Central

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co- cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes. PMID:7175438

1982-01-01

79

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

PubMed Central

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

Xiao, Yin; Mareddy, Shobha; Crawford, Ross

2010-01-01

80

Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro.  

PubMed

Dendritic cells (DC) can be generated from mouse bone marrow (BM) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bacterial stimuli such as endotoxin / lipopolysaccharide (LPS) can induce their final maturation. When BM-DC cultures were treated at day 6 or later with LPS, this final maturation was induced in vitro within 24 h. Such mature DC exhibited high levels of surface MHC II molecules and potent T cell sensitizing, but reduced endocytosis capacity. In contrast, immature DC express only few MHC II molecules and are weak T cell stimulators but highly endocytic. When BM-DC cultures in GM-CSF were treated with 1 microg / ml LPS at day 0 of the culture or throughout the culture, only immature DC developed as defined by phenotype (MHC II low) and function (high endocytosis, weak primary mixed lymphocyte reaction). Those early LPS-treated immature DC induced alloantigen-specific anergy of CD4(+) T cells in vitro. These findings might contribute to the understanding of reduced T cell immunity in the course of septic shock and find application in DC-mediated tolerogenic immunotherapy strategies. PMID:10760792

Lutz, M B; Kukutsch, N A; Menges, M; Rössner, S; Schuler, G

2000-04-01

81

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. PMID:24947048

2014-01-01

82

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

Microsoft Academic Search

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

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

1996-01-01

83

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

PubMed Central

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

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

2014-01-01

84

Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner  

Microsoft Academic Search

Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with a capacity to form bone tissue in vivo, and to differentiate into the osteoblastic lineage in vitro. Drawing on evidence that bone is mechanosensitive and mechanical stimuli are anabolic, we postulate that proliferation and osteoblastic differentiation of BMSCs may be stimulated by mechanical forces. In this study, BMSCs cultured in

Michelle R. Kreke; William R. Huckle; Aaron S. Goldstein

2005-01-01

85

Time-related changes in expression of collagen types I and III and of tenascin-C in rat bone mesenchymal stem cells under co-culture with ligament fibroblasts or uniaxial stretching  

Microsoft Academic Search

Adult bone-marrow-derived mesenchymal stroma cells (BMSC) seem to be a potential cell source for tissue engineering of the\\u000a ligament. The objective of this work was to study the time-related changes in mRNA expression and protein levels of collagen\\u000a types I and III and of tenascin-C in BMSC under co-culture with fibroblasts or under a uniaxial cyclic condition. Rat BMSC\\u000a harvested

Lei Zhang; Nguyen Tran; Huai-Qing Chen; Cyril J.-F. Kahn; Sophie Marchal; Frederique Groubatch; Xiong Wang

2008-01-01

86

Basics of Cell Culture  

NSDL National Science Digital Library

These manuals are used in the Stem Cell Culture Course at City College of San Francisco. This course is about general mammalian cell culture techniques but includes a laboratory exercise using stem cells (takes 3 weeks to complete). The course is taught to high school students but the materials are also used for college students. Laboratory exercises provide instruction in basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells. Photographs and explanations of common equipment (laminar flow hood, inverted microscope, etc.) and reagents are provided. Laboratory exercises include the following: Basic Aseptic Technique; Media Preparation; Plating cells from frozen stock; Cell counting and plating; Survival assay (UV); Live Cell Identification; Transfection; Freezing cells; Stem cell differentiation. A student lab manual and an instructor manual are provided.

Afshar, Golnar

87

Special stem cells for bone.  

PubMed

Mesenchymal stem cells (MSCs) are multipotential in vitro, but their endogenous properties are poorly defined. In this issue of Cell Stem Cell, Park et al. (2012) report that an MSC-like, osteolineage-directed Mx1+ population generates new osteoblasts at sites of bone damage, suggesting its potential for skeletal repair and regeneration. PMID:22385649

Zaidi, Mone; Sun, Li; Blair, Harry C

2012-03-01

88

Giant cell tumor of bone.  

PubMed

Giant cell tumor (GCT) of bone is one type of giant cell-rich lesion of bone. This benign mesenchymal tumor has characteristic multinuclear giant cells. Mononuclear stromal cells are the physiologically active and diagnostic cell type. Most GCTs are located in the epiphyseal regions of long bones. The axial skeleton-primarily the sacrum-is a secondary site of involvement. Most patients present with pain, swelling, joint effusion, and disability in the third and fourth decades of life. Imaging studies are important for tumor staging and radiographic grading. Typically, these clinically active but slow-growing tumors are confined to bone, with relatively well-defined radiographic borders. Monostotic disease is most common. Metastatic spread to the lungs is rare. Extended intralesional curettage with or without adjuvant therapy is the primary treatment choice. Local recurrence is seen in ? 20% of cases, and a second local intralesional procedure is typically sufficient in cases that are detected early. Medical therapies include diphosphonates and denosumab. Denosumab has been approved for use in osteoporosis as well as breast and prostate cancer metastatic to bone. Medical therapy and radiotherapy can alter the management of GCT of bone, especially in multifocal disease, local recurrences, and bulky central/axial disease. PMID:23378375

Raskin, Kevin A; Schwab, Joseph H; Mankin, Henry J; Springfield, Dempsey S; Hornicek, Francis J

2013-02-01

89

Evidence for Structured Variation in Self-Renewal Capacity within Long-Term Bone Marrow Cultures  

Microsoft Academic Search

Bone marrow pluripotent stem cells (CFUs) demonstrate capacity for both proliferation and differentiation. The proliferative capacity of CFUs has been measured by serial transplantability and by the Rs, a measurement of CFU production in a single 14-day transfer. In the present study, the self-renewal capacity of both adherent and nonadherent CFUs from long-term bone marrow cultures was measured. Culture conditions

Peter Mauch; Joel S. Greenberger; Leslie Botnick; Eileen Hannon; Samuel Hellman

1980-01-01

90

Mammalian Cell Culture  

NSDL National Science Digital Library

This "Course-in-a-Box" from Bio-Link is a good starting point for instructors to develop a course on how to maintain mammalian cells in culture. Students will learn "basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells." Laboratories include Basic Aseptic Technique, Media Preparation, and Plating Cells from Frozen Stock. Materials include an Instructor Laboratory Manual, Student Laboratory Manual, Problem Sets, and Quizzes. A free login is required to access the materials.

91

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

92

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

93

Bone marrow stromal cell assays – in vitro and in vivo  

PubMed Central

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

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

2014-01-01

94

Human preleukaemia cell culture studies in sideroblastic anaemia  

Microsoft Academic Search

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,

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

1976-01-01

95

Carbonic Anhydrase and Bone Remodeling: Sulfonamide Inhibition of Bone Resorption in Organ Culture  

Microsoft Academic Search

Five sulfonamide inhibitors of carbonic anhydrase inhibited parathyroid hormone-induced resorption of bone in organ culture. The relative activities of the sulfonamides as resorption inhibitors were such as to suggest the presence of a functional carbonic anhydrase system in bone linked to the mechanism of bone resorption.

Cedric Minkin; Joan M. Jennings

1972-01-01

96

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

PubMed Central

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

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

2014-01-01

97

Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent  

E-print Network

- sue-remodeling process in bone and are potential tools for tissue engineering and cell-based therapies and maintenance of progenitor cells during in vitro culture. Stem Cells 2004;22:39-50 STEM CELLS 2004;22:39-50 www.StemCells engineering and cell-based therapies for skeletal pathologies. Osteoblast activities including bone matrix

Zandstra, Peter W.

98

Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis  

Microsoft Academic Search

The nonhematopoietic stromal cells of the bone marrow are critical for the development of hematopoietic stem cells into functionally competent blood cells. This study addresses the question of whether bone marrow stromal cell cultures in the Dexter system propagate multiple different mesenchymal stromal cell types or one stromal cell type that expresses multiple phenotypes. Results show that isolated single stromal

Beerelli Seshi; Sanjay Kumar; Debra King

2003-01-01

99

Leukemia cells induce changes in human bone marrow stromal cells  

PubMed Central

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

2013-01-01

100

Cell-culture bioreactors  

SciTech Connect

When contrasted with microbial fermentation, the characteristics having a bearing on the design and operation of cell-culture bioreactors are fragility, steam sensitivity and anchorage requirements of cells, heat lability and foaming of proteins and other components of cell culture media. Design details of agitation and gas supply, bearings, seals and drives, foam control and sterilization, temperature, oxygen and pH control, water, air and gas purification, liquid feeding and level control, gas exhaust analysis and disposal, handling of liquid effluent and bioreactor installation and scale up are given.

Beck, C.; Stiefel, H.; Stinnett, T.

1987-02-16

101

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

PubMed Central

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

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

2011-01-01

102

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

103

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. PMID:24198436

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

2013-01-01

104

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

105

CONTRACTION AND ORGANIZATION OF COLLAGEN GELS BY CELLS CULTURED FROM PERIODONTAL LIGAMENT, GINGIVA AND BONE SUGGEST FUNCTIONAL DIFFERENCES BETWEEN CELL TYPES  

Microsoft Academic Search

SUMMARY Monkey periodontal ligament fibroblasts (MPLF cells), human gingival fibroblasts (HGF cells), rat embryonic calvaria cells (REC cells), porcine periodontal ligament epithelial cells (PPLE cells) and rat osteosarcoma 17\\/2 cells (ROS cells) were incorporated into 3-dimensional collagen gels plated in 60 mm Petri dishes in order: first, to measure the capacity of these cell types to contract; second, to investigate

C. G. BELLOWS; A. H. MELCHER; J. E. AUBIN

1981-01-01

106

Bone nodule formation of Mg63 cells is increased by the interplay of signaling pathways cultured on vitamin D3?entrapped calcium phosphate films  

Microsoft Academic Search

Since vitamin D3 is an important regulator of osteoblastic differentiation, a presently?established vitamin D3?entrapped calcium phosphate film (VCPF) was evaluated for hard tissue engineering. The entrapped vitamin D3 more rapidly induced bone nodule formation. To characterize the cellular events leading to regulations including faster differentiation, signal transduction pathways were investigated in osteoblastic MG63 cells at a molecular level. Major signaling

Yong Seok Choi; Yoon Jung Hong; Jung Hur; Mee Young Kim; Jae Young Jung; Sunjoo Jeong

2009-01-01

107

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

E-print Network

Bone marrow: all the cells of the immune system are derived from stem cells in the bone marrow. The bone marrow is the site of origin of red blood cells, white cells (including lymphocytes and macrophages) and platelets. Thymus: in the thymus gland lymphoid cells undergo a process of maturation

Morante, Silvia

108

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

Microsoft Academic Search

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

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

2011-01-01

109

Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells  

PubMed Central

Eosinophils are produced in the bone marrow from CD34+ eosinophil lineage–committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage–committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage–committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage–committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage–committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5. PMID:25551463

Schollaert, Kaila L.; Stephens, Michael R.; Gray, Jerilyn K.; Fulkerson, Patricia C.

2014-01-01

110

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

111

Perfusion Based Cell Culture Chips  

NASA Astrophysics Data System (ADS)

Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.

Heiskanen, A.; Emnéus, J.; Dufva, M.

112

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

113

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

Microsoft Academic Search

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

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

2006-01-01

114

Bone marrow derived stem cells in joint and bone diseases: a concise review.  

PubMed

Stem cells have huge applications in the field of tissue engineering and regenerative medicine. Their use is currently not restricted to the life-threatening diseases but also extended to disorders involving the structural tissues, which may not jeopardize the patients' life, but certainly influence their quality of life. In fact, a particularly popular line of research is represented by the regeneration of bone and cartilage tissues to treat various orthopaedic disorders. Most of these pioneering research lines that aim to create new treatments for diseases that currently have limited therapies are still in the bench of the researchers. However, in recent years, several clinical trials have been started with satisfactory and encouraging results. This article aims to review the concept of stem cells and their characterization in terms of site of residence, differentiation potential and therapeutic prospective. In fact, while only the bone marrow was initially considered as a "reservoir" of this cell population, later, adipose tissue and muscle tissue have provided a considerable amount of cells available for multiple differentiation. In reality, recently, the so-called "stem cell niche" was identified as the perivascular space, recognizing these cells as almost ubiquitous. In the field of bone and joint diseases, their potential to differentiate into multiple cell lines makes their application ideally immediate through three main modalities: (1) cells selected by withdrawal from bone marrow, subsequent culture in the laboratory, and ultimately transplant at the site of injury; (2) bone marrow aspirate, concentrated and directly implanted into the injury site; (3) systemic mobilization of stem cells and other bone marrow precursors by the use of growth factors. The use of this cell population in joint and bone disease will be addressed and discussed, analysing both the clinical outcomes but also the basic research background, which has justified their use for the treatment of bone, cartilage and meniscus tissues. PMID:25005462

Marmotti, Antonio; de Girolamo, Laura; Bonasia, Davide Edoardo; Bruzzone, Matteo; Mattia, Silvia; Rossi, Roberto; Montaruli, Angela; Dettoni, Federico; Castoldi, Filippo; Peretti, Giuseppe

2014-09-01

115

Culture of organized cell communities  

Microsoft Academic Search

Cells cultured in vitro will tend to retain their differentiated phenotype under conditions that resemble their natural in vivo environment, for example, when cultured on polymer scaffolds in tissue culture bioreactors. In this chapter, we define organized cell communities as three-dimensional in vitro grown cell–polymer constructs that display important structural and functional features of the natural tissue. We review representative

Lisa E. Freed; Gordana Vunjak-Novakovic

1998-01-01

116

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

117

In vitro proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells cultured with hardystonite (Ca2ZnSi 2O7) and {beta}-TCP ceramics.  

PubMed

The effects of hardystonite (Ca(2)ZnSi(2)O(7), CSZn) and tricalcium phosphate (beta-TCP) on the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) were compared by directly culturing MSCs on ceramic disks (contact mode) or separately culturing cells with ceramic disks (non-contact mode). In non-contact mode, the CSZn ceramic supported MSC proliferation more strongly than did the beta-TCP ceramic. However, in contact mode, the MSCs proliferated more quickly on the beta-TCP ceramic than they did on the CSZn ceramic. Alkaline phosphatase (ALP) staining and osteogenic gene expression analysis showed that the CSZn and beta-TCP ceramics had significant effects on the promotion of the osteogenic differentiation of MSCs in both non-contact and contact mode. Furthermore, in contact mode, the CSZn disk promoted the osteogenic differentiation of MSCs more strongly than did the beta-TCP disks. Even without the induction of dexamethasone and beta-glycerophosphate, CSZn stimulated the osteogenic differentiation of MSCs. These results suggest that CSZn ceramic would be a useful candidate material for bone regeneration and hard tissue engineering. PMID:19726532

Lu, Hongxu; Kawazoe, Naoki; Tateishi, Tetsuya; Chen, Guoping; Jin, Xiaogang; Chang, Jiang

2010-07-01

118

Bone disorders in sickle-cell disease  

Microsoft Academic Search

Sickle cell disease involves many organs but musculo-skeletal problems present most often with bone pain, the most common reason for admission to hospital. Two separate pathological abnormalities cause these lesions. Sickling of the red cells produces thromboembolic infarcts in bone leading to pain, crises and sometimes osteomyelitis; increased destruction of sickle red cells produces haemolysis, an increase in erythroblastic activity

O. Onuba

1993-01-01

119

Bone regeneration at dental implant sites with suspended stem cells.  

PubMed

During the maintenance of bone marrow-derived mesenchymal stem cells (BMMSCs), suspended cells are discarded normally. We noted the osteogenic potential of these cells to be like that of anchorage-dependent BMMSCs. Therefore, we characterized suspended BMMSCs from rabbit bone marrow by bioengineering and applied the suspended BMMSCs to double-canaled dental implants inserted into rabbits. After primary isolation of BMMSCs, we collected the suspended cells during primary culture on the third day. The cells were transferred and maintained on an extracellular-matrix-coated culture plate. The cells were characterized and compared with BMMSCs by colony-forming-unit fibroblast (CFU-f) and cell proliferation assay, fluorescence-activated cell sorter (FACS), in vitro multipotency, and reverse transcription polymerase chain reaction (RT-PCR). We also analyzed the osteogenic potential of cells mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and transplanted into immunocompromised mice. We compared the viability and proliferation of the suspended BMMSCs and BMMSCs on the titanium implant surface and observed cell morphology. Then, the cells mixed with HA/TCP were applied to the double-canaled implants during installation into rabbit tibia. Four weeks later, we analyzed bone formation inside the canal by histomorphometry. The suspended cells showed higher CFU-f on the extracellular matrix (ECM)-coated culture plate and similar results of proliferation capacity compared with BMMSCs. The cells also showed osteogenic, adipogenic, and chondrogenic ability. The suspended cells showed levels of attachment survival and proliferation on the surfaces of titanium implant discs to be higher than or similar to those of BMMSCs. The suspended cells as well as BMMSCs showed stronger bone formation ability in both upper and lower canals of the implants compared with controls on double-canaled implants inserted into rabbit tibia. In this study, we showed that suspended cells after primary BMMSC isolation have bone regeneration capacity like that of BMMSCs, not only in vitro but also in vivo. ECM was valuable for propagation of MSCs for cell-based bone regeneration. Therefore, the suspended cells could also be useful tools for bone regeneration after implant surgery. PMID:25183420

Zheng, R C; Park, Y K; Cho, J J; Kim, S K; Heo, S J; Koak, J Y; Lee, J H

2014-10-01

120

Evidence for the expression of estrogen receptors in osteogenic cells isolated from hen medullary bone.  

PubMed

Medullary bone is a unique tissue in female birds and forms in the cavity of long bones. This bone displays rapid remodeling in response to circulating estrogen levels, suggesting that the osteoblasts in this bone are highly sensitive to estrogen. The present study examined expression of two estrogen receptor (ER) mRNAs in osteogenic cells of medullary bone of white Leghorn hens in vitro. At day 3, isolated cells from the hen medullary bone expressed alkaline phosphatase activity. Using immunocytochemistry, ER protein was demonstrated in the nuclei of these cells. RT-PCR analysis revealed that ER-alpha mRNA was constantly expressed from day 3 to day 15 of culture, while ER-beta mRNA was not detected throughout the culture period. These results indicate that estrogen may act via ER-alpha, but not ER-beta, on osteogenic cells of the avian medullary bone. PMID:18835015

Hiyama, Shinji; Sugiyama, Toshie; Kusuhara, Seiji; Uchida, Takashi

2009-01-01

121

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

NASA Astrophysics Data System (ADS)

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

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

122

A Non-Contact Suspension Culture Approach to the Culture of Osteogenic Cells Derived from a  

E-print Network

: mesenchymal stem cells; suspension culture; osteogenesis Introduction Adult bone marrow (BM) is the most common tissue used as a source of mesenchymal stromal cells (MSCs) and multiple studies have identified markers that can be used to identify BM-derived MSCs as mesenchymal stem cells, as described by Dominici

Zandstra, Peter W.

123

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

124

The effect of Gu-Sui-Bu (Drynaria fortunei J. Sm) on bone cell activities.  

PubMed

In the traditional Chinese medicine, Gu-Sui-Bu [Drynaria fortunei (kunze) J. Sm] has been reported as a good enhancer for bone healing. In this experiment, we investigate the biochemical effects of this traditional Chinese medicine on the bone cells culture. Different concentrations of crude extract of Gu-Sui-Bu were added to rat bone cells culture. The mitochondria activity of the bone cells after exposure was determined by colorimetric assay. Biochemical markers such as alkaline phosphatase (ALP), acid phosphatase (ACP) titer, prostaglandin E2 (PGE2) titer and the expression of both osteopontin and osteonectin mRNA were evaluated. The effect on the osteoclasts differentiation was evaluated by tartrate-resistant acid phosphatase (TRAP) stain. The most effective concentration of Gu-Sui-Bu on bone cells was 1 mg/ml. The addition of 1 mg/ml Gu-Sui-Bu to bone cells culture for 7 days can statistically increase the intracellular ALP amount; while the ACP and PGE2 amount in culture medium were significantly increased. In Northern blot analysis, the expression of both osteopontin and osteonectin mRNA were down-regulated after adding Gu-Sui-Bu into bone cells culture. The formation of multi-nucleated osteoclasts was more active than that of the control group, but no giant osteoclasts formation was observed. In this study, we demonstrated that Gu-Sui-Bu has potential effects on the bone cells culture. One of the major effects of Gu-Sui-Bu on the bone cells is probably mediated by its effect on the osteclasts activities. Continued and advanced study on the alterations in gene expression of bone cells by Chinese medicines will provide a basis for understanding the observed bone cell responses to various pharmacological interventions. PMID:12099280

Sun, Jui-Sheng; Lin, Chun-Yu; Dong, Guo-Chung; Sheu, Shiow-Yunn; Lin, Feng-Huei; Chen, Li-Ting; Wang, Yng-Jiin

2002-08-01

125

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. PMID:23597232

2013-01-01

126

Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells  

PubMed Central

Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws. PMID:20811069

Aghaloo, T.L.; Chaichanasakul, T.; Bezouglaia, O.; Kang, B.; Franco, R.; Dry, S.M.; Atti, E.; Tetradis, S.

2010-01-01

127

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

PubMed

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

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

2010-12-15

128

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

129

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

PubMed Central

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

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

2013-01-01

130

Human long-term culture initiating cell assay.  

PubMed

The long-term culture initiating cell (LTC-IC) assay, founded on the bone marrow long-term culture (LTC) system, measures primitive hematopoietic stem cells (termed LTC-IC) based on their capacity to produce myeloid progeny for at least 5 weeks. Adaptations of the LTC system including the use of stromal cell lines, application of limiting dilution analysis, and estimation of average hematopoietic progenitor output per LTC-IC under defined conditions have made it possible to accurately determine LTC-IC content in minimally separated and highly purified cell populations from human hematopoietic tissue sources such as bone marrow, peripheral blood, cord blood, fetal liver as well as cord blood and mobilized peripheral blood. Methodologies for measuring human LTC-IC using bulk cultures, limiting dilution analysis, and single cell cultures are described. PMID:23179836

Liu, Min; Miller, Cindy L; Eaves, Connie J

2013-01-01

131

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

PubMed

Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd. PMID:23166000

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

2015-02-01

132

Bone Marrow Stromal Cells Generate Muscle Cells and  

E-print Network

12/14/09 1 Bone Marrow Stromal Cells Generate Muscle Cells and Repair Muscle Degeneration By Stephanie Aracena & Roynan Krebs Bone Marrow Stromal Cells What are they? Where are they located? Why not stem cells? Small populations Ethical reasons Histocompatibility Difficult to get #12;12/14/09 2

Devoto, Stephen H.

133

Culture of Human Leukaemia Cells  

Microsoft Academic Search

THIS communication describes the culture of four additional cell lines derived from the buffy coats of patients with leukaemia. Iwakata and Grace1 provided key information for culturing leukaemia cells and reported the establishment of a cell line, R.P.M.I. No. 6410. Fifteen cell lines were subsequently derived from the buffy coats of four patients with acute and chronic myelocytic leukaemia and

G. E. Moore; E. Ito; P. Citron

1966-01-01

134

Primary Cilia Exist in a Small Fraction of Cells in Trabecular Bone and Marrow.  

PubMed

Primary cilia are potent mechanical and chemical sensory organelles in cells of bone lineage in tissue culture. Cell culture experiments suggest that primary cilia sense fluid flow and this stimulus is translated through biochemical signaling into an osteogenic response in bone cells. Moreover, in vivo, primary cilia knockout in bone cells attenuates bone formation in response to loading. However, understanding the role of the primary cilium in bone mechanotransduction requires knowledge of its incidence and location in vivo. We used immunohistochemistry to quantify the number of cells with primary cilia within the trabecular bone tissue and the enclosed marrow of ovine cervical vertebrae. Primary cilia were identified in osteocytes, bone lining cells, and in cells within the marrow, but were present in only a small fraction of cells. Approximately 4 % of osteocytes and 4.6 % of bone lining cells expressed primary cilia. Within the marrow space, only approximately 1 % of cells presented primary cilia. The low incidence of primary cilia may indicate that cilia either function as mechanosensors in a selected number of cells, function in concert with other mechanosensing mechanisms, or that the role of primary cilia in mechanosensing is secondary to its role in chemosensing or cellular attachment. PMID:25398598

Coughlin, Thomas R; Voisin, Muriel; Schaffler, Mitchell B; Niebur, Glen L; McNamara, Laoise M

2014-11-15

135

Bone marrow mesenchymal stem cells and TGF-? signaling in bone remodeling  

PubMed Central

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

Crane, Janet L.; Cao, Xu

2014-01-01

136

Influence of cassette design on three-dimensional perfusion culture of artificial bone.  

PubMed

Media perfusion is often required to maintain cell viability within topographically complex 3-dimensional scaffold cultures. Osteoblast-seeded scaffolds for bone regeneration require robust cell proliferation and survival both within the scaffold and over the exterior for optimal osteogenic capacity. Conventional press-fitting cassettes ensure internal fluid flow through the scaffold but may restrict external flow around the scaffold, resulting in a barren (cell-free) external scaffold surface. In this study, we aimed to solve this problem by modifying the cassette structure to enhance external flow in an oscillatory perfusion culture system. Mouse osteoblast-like MC 3T3-E1 cells were seeded in porous ceramic scaffolds and incubated for 3 days either under static culture conditions or in an oscillatory perfusion bioreactor. Scaffolds were held in the bioreactor with either conventional press-fitting cassettes or cassettes with rings to separate the scaffold exterior from the internal cassette wall. The external surfaces of scaffolds maintained under static conditions were well seeded, but cells failed to grow deeply into the core, reflecting poor internal chemotransport. Alternatively, scaffolds cultured by perfusion with press-fitting cassettes had poor cell viability at the cassette-external scaffold surface interface, but cells were widely distributed within the scaffold core. Scaffolds cultured using the modified cassettes with 1 or 2 rings exhibited uniformly distributed living cells throughout the internal pores and over the entire external surface, possibly because of the improved medium flow over the scaffold surface. This modified oscillatory perfusion culture system may facilitate the production of engineered bone with superior osteogenic capacity for grafting. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 84-91, 2015. PMID:24764314

Du, Dajiang; Ushida, Takashi; Furukawa, Katsuko S

2015-01-01

137

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

138

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

Microsoft Academic Search

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

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

2003-01-01

139

Expansion of hematopoietic progenitor cell populations in stirred suspension bioreactors of normal human bone marrow cells.  

PubMed

We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells. Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors, detected as long-term culture-initiating cells (LTC-IC), could be achieved as well in stirred suspension cultures as in static cultures. Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment. Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC, CFC and total cell numbers in stirred cultures. Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold, respectively, above input values. Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer. Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations. In addition, stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms. PMID:7521647

Zandstra, P W; Eaves, C J; Piret, J M

1994-09-01

140

Microenvironmental niches in the bone marrow required for B-cell development  

Microsoft Academic Search

B-cell development is known to occur in a complex bone-marrow microenvironment but its functional organization remains unclear. It is thought that bone-marrow stromal cells create distinct microenvironments, known as niches, that provide support for haematopoiesis and B-cell development. Although it has been more than 20 years since the development of a culture system that allows the growth of B-cell progenitors

Takashi Nagasawa

2006-01-01

141

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. 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

142

Restricted and coordinated expression of ? 3-integrin and bone sialoprotein during cultured osteoblast differentiation  

Microsoft Academic Search

In this study, the expression of ?3-integrin was examined in relationship to the restricted expression of bone sialoprotein (BSP). Immunohistochemical analysis indicated that the ?v?3-integrin was coincident and proximal to BSP expression in the fetal mandible bovine osteoblast culture model. ?v?3-integrin expression was expressed predominantly in a region proximal to, but not including, the substrate adherent cells. In comparison, the

G. B Schneider; S. W Whitson; L. F Cooper

1999-01-01

143

Cellular and molecular phenotypes of osteogenic cells isolated from the medullary bone of the hen in vitro.  

PubMed

In this study, cells isolated from hen medullary bone were cultured to examine their matrix formation. Furthermore, we compared medullary bone cells with rat bone marrow cells regarding the temporal changes in osteoblast developmental markers. Medullary bone cells were positive for alkaline phosphatase (ALP) activity and formed bone nodules, apparent with Alcian blue and von Kossa staining. The intensity of these stains became stronger with the maturation of those bone nodules. In this developmental process, the expression patterns of osteoblast phenotypes of medullary bone cells differed from those of rat bone marrow cells. ALP mRNA was expressed at the maximum level in the proliferation stage and gradually decreased in medullary bone cells, but that expression showed the opposite pattern in rat bone marrow cells. Medullary bone cells strongly expressed two non-collagenous protein mRNAs from the early stages, but the expression of these mRNAs in rat bone marrow cells increased only in the later stages. These results suggest that the features of medullary bone osteoblasts differ from those of mammalian osteoblasts and are reflected in the characteristics of medullary bone in vivo. PMID:16236535

Hiyama, Shinji; Sugiyama, Toshie; Kusuhara, Seiji; Uchida, Takashi

2005-12-01

144

Curcumin analog UBS109 prevents bone marrow osteoblastogenesis and osteoclastogenesis disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro.  

PubMed

UBS109 is a curcumin analog that possesses antitumor properties has been shown to stimulate osteoblastogenesis and suppress osteoclastogenesis in vitro. This study was undertaken to determine whether UBS109 might alleviate the inhibitory activity of breast cancer cells on osteoblastic mineralization and stimulatory effects on osteoclastogenesis. Mouse bone marrow cells were cocultured with breast cancer MDA-MB-231 bone metastatic cells in vitro. UBS109 stimulated osteoblastic mineralization and suppressed adipogenesis and osteoclastogenesis in bone marrow culture. Coculture with MDA-MB-231 cells suppressed osteoblastic mineralization and enhanced osteoclastogenesis in bone marrow culture. Effects that were reserved by UBS109 (50-200 nM). Mineralization in preosteoblastic MC3T3-E1 cells was suppressed by coculture with MDA-MB-231 cells, while MDA-MB-231 cells did not have effects on osteoclastogenesis of RAW267.4 cells in vitro. UBS109 (500 nM) revealed toxic effects on MDA-MB-231 bone metastatic cells. This study demonstrates that UBS109, which is an antitumor agent, reveals restorative effects on bone marrow cell differentiation disordered by coculture with breast cancer MDA-MB-231 bone metastatic cells in vitro. This in vitro model may be a useful tool to evaluate the mechanism of breast cancer cell bone metastasis. PMID:25416449

Yamaguchi, Masayoshi; Zhu, Shijun; Weitzmann, M Neale; Snyder, James P; Shoji, Mamoru

2015-03-01

145

Differential marker expression by cultures rich in mesenchymal stem cells  

PubMed Central

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

2013-01-01

146

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

147

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

PubMed Central

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

Roato, Ilaria

2013-01-01

148

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

PubMed Central

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

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

2015-01-01

149

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

NASA Technical Reports Server (NTRS)

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

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

1997-01-01

150

BMP3 Suppresses Osteoblast Differentiation of Bone Marrow Stromal Cells via Interaction with Acvr2b  

PubMed Central

Enhancing bone morphogenetic protein (BMP) signaling increases bone formation in a variety of settings that target bone repair. However, the role of BMP in the maintenance of adult bone mass is not well understood. Targeted disruption of BMP3 in mice results in increased trabecular bone formation, whereas transgenic overexpression of BMP3 in skeletal cells leads to spontaneous fracture, consistent with BMP3 having a negative role in bone mass regulation. Here we investigate the importance of BMP3 as a mediator of BMP signaling in the adult skeleton. We find that osteoblasts (OBL) and osteocytes are the source of BMP3 in adult bone. Using in vitro cultures of primary bone marrow stromal cells, we show that overexpression of BMP3 suppresses OBL differentiation, whereas loss of BMP3 increases colony-forming unit fibroblasts and colony-forming unit OBL. The ability of BMP3 to affect OBL differentiation is due to its interaction with activin receptor type 2b (Acvr2b) because knockdown of endogenous Acvr2b in bone marrow stromal cells reduces the suppressive effect of BMP3 on OBL differentiation. These findings best fit a model in which BMP3, produced by mature bone cells, acts to reduce BMP signaling through Acvr2b in skeletal progenitor cells, limiting their differentiation to mature OBL. Our data further support the idea that endogenous BMPs have a physiological role in regulating adult bone mass. PMID:22074949

Kokabu, Shoichiro; Cox, Karen; Lowery, Jonathan; Tsuji, Kunikazu; Raz, Regina; Economides, Aris; Katagiri, Takenobu; Rosen, Vicki

2012-01-01

151

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

SciTech Connect

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

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

2012-03-23

152

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

153

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

PubMed Central

The tendon/ligament-to-bone interface has a complex organization to enable transfer of forces through the tendon/ligament to the bone. The purpose of this study is to create a co-culture environment enabling a tissue-specific tendon region and tissue-specific bone region on a degradable scaffold, using NIH 3T3 fibroblast–deposited extracellular matrix and MC 3T3 osteoblast–deposited extracellular matrix, respectively. Before full characterization of the deposited extracellular matrix coating can be analyzed, co-culture parameters including culture medium and seeding technique should be addressed. An appropriate medium formulation was developed to reduce fibroblast to osteoblast mineralization by adjusting beta-glycerophosphate concentrations. Standard growth medium with fetal bovine serum + 3 mM beta-glycerophosphate + 25 µg/mL ascorbic acid was found to be the most suitable formulation evaluated in these study conditions. Seeding and cell migration studies of co-cultured fibroblast- and osteoblast-specific scaffolds were performed to identify whether tissue regions could be created on the scaffold. Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding. Finally, a preliminary analysis of basic extracellular matrix components was measured in the fibroblast, osteoblast, and transition regions. Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions. In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold. PMID:25383167

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

2014-01-01

154

Repair of bone defect using bone marrow cells and demineralized bone matrix supplemented with polymeric materials.  

PubMed

We present a novel, reverse thermo-responsive (RTR) polymeric osteogenic composite comprising demineralized bone matrix (DBM) and unmanipulated bone marrow cells (BMC) for repair of bone defects. The polymers investigated were low viscosity aqueous solutions at ambient temperature, which gel once they heat up and reach body temperature. Our goal to supplement DBM-BMC composite with RTR polymers displaying superior rheological properties, was to improve graft integrity and stability, during tissue regeneration. The osteogenic composite when implanted under kidney capsule of mice, proved to be biocompatible and biodegradable, with no residual polymer detected in the newly formed osteohematopoietic site. Implantation of the osteogenic composite into a large area of missing area of parietal bone of the skull of rats, resulted in an extensive remodeling of DBM particles, fully reconstituted hematopoietic microenvironment and well integrated normal flat bone within thirty days. The quality and shape of the newly created bone were comparable to the original bone and neither local or systemic inflammatory reactions nor fibrosis at the junction of the new and old calvarium could be documented. Furthermore, combined laser capture microdissection (LCM) technique and PCR analysis of male BMC in female rats confirmed the presence of male derived cells captured from the repaired/ regenerated flat bone defect. The use of active self sufficient osteogenic DBM-BMC composite supported by a viscous polymeric scaffold for purposive local hard tissue formation, may have a significant potential in enhancement of bone regeneration and repair following trauma, degenerative or inflamatory lesion, iatrogenic interventions and cosmetic indications. PMID:19807659

Kurkalli, Basan Gowda S; Gurevitch, Olga; Sosnik, Alejandro; Cohn, Daniel; Slavin, Shimon

2010-03-01

155

Long term culture of tumour-specific cytotoxic T cells  

Microsoft Academic Search

MANY investigators have been successful in the maintenance of long term tissue culture of human bone marrow-derived (B) cells. These cell lines have been established from both normal subjects1 and from patients with lymphoproliferative disorders2. In most cases, long term B-cell lines have been shown to harbour the Epstein-Barr virus genome which some investigators feel is required for establishment and

Steven Gillis; Kendall A. Smith

1977-01-01

156

Regulation of bone-related genes expression by bone-like apatite in MC3T3-E1 cells.  

PubMed

Bone-like apatite on HA/TCP ceramics sintered at 1,100 degrees C (HT1) and 1,200 degrees C (HT2) could be obtained via immersing substrates into simulated body fluid (SBF) for 3 days. When MC3T3-E1 preosteoblastic cells cultured on the surface of the bone-like apatite for 3 days, SEM observations revealed cell membrane features with secreted crystals very similar to in vivo bone formation during intramembranous ossification with a direct bone apposition on the ceramics. According to semi-quantitative RT-PCR method, mRNA expressions of osteocalcin (marker of late-stage differentiation) and type 1 collagen were increased in cultures with HT1S and HT2S when compared to HT1 and HT2 after cultured for 6 days. The results indicated that bone-like apatite had the ability to support the growth of osteoblast-like cells in vitro and to promote osteoblast differentiation by stimulating the expression of major phenotypic markers. Taken together, our findings will be helpful in understanding the mechanism of osteoinductivity of calcium phosphate ceramics and in constructing more appropriate biomimetic substrate. PMID:17597361

Tan, Y F; Hong, S F; Wang, X L; Lu, J; Wang, H; Zhang, X D

2007-11-01

157

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. PMID:22447716

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

2012-01-01

158

Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells  

PubMed Central

Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1+, a myeloid cell marker, and found predominantly in the bone marrow; in parenchymal tissues. Surprisingly, fused cells were most abundant in the kidney, Peyer’s patches, and cardiac tissue. In contrast, after cell fusion with embryonic stem cells, bone marrow cells were reprogrammed into new tetraploid pluripotent stem cells that successfully differentiated into beating cardiomyocytes. Together, these data suggest that cell fusion is ubiquitous after cellular transplants and that the subsequent sharing of genetic material between the fusion partners affects cellular survival and function. Fusion between tumor cells and bone marrow cells could have consequences for tumor malignancy.—Bonde, S., Pedram, M., Stultz, R., Zavazava, N. Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells. PMID:19762558

Bonde, Sabrina; Pedram, Mehrdad; Stultz, Ryan; Zavazava, Nicholas

2010-01-01

159

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

160

Inhibitory effects of osteoblasts and increased bone formation on myeloma in novel culture systems and a myelomatous mouse model  

PubMed Central

Background and Objectives Multiple myeloma (MM) growth in the bone marrow is associated with increased osteoclast activity and a reduced number of osteoblasts. Experimental studies suggest that bone disease drives the progression of MM. Whereas those studies focused on the critical role of myeloma-induced osteoclastogenesis in disease progression, little is known about the impact of osteoblasts and increased bone formation on MM. Design and Methods We investigated the effect of isolated osteoblasts and osteoclasts on survival and proliferation of primary MM plasma cells (PC) in co-cultures and triple-cultures, and tested the effect of mesenchymal stem cells (MSC) on bone mineral density and MM growth in myelomatous human bones of SCID-hu mice. Results Whereas osteoclasts promoted survival and proliferation of MM PC, osteoblasts supported or inhibited MM PC, depending on the source of the MM cells. In triple-cultures osteoblasts attenuated the effect of osteoclasts on MM PC in 18 of 24 experiments. The anti-MM response to osteoblasts correlated with advanced clinical stage. Injection of MSC into myelomatous bones resulted in marked inhibition of tumor growth in three of nine experiments and stabilization of disease in two additional experiments. The anti-MM response of MSC was associated with increased human bone mineral density. Immunohistochemical analysis indicated that the MSC were well engrafted and, in responding mice, differentiated into osteogenic cells. Interpretation and Conclusions MM PC from the majority of patients are susceptible to growth inhibition by osteoblasts; however, growth of MM PC from certain patients is accelerated by osteoblasts. In vivo, increased bone formation is associated with reduced myeloma burden. PMID:16461303

Yaccoby, Shmuel; Wezeman, Michele J.; Zangari, Maurizio; Walker, Ronald; Cottler-Fox, Michele; Gaddy, Danna; Ling, Wen; Saha, Rinku; Barlogie, Bart; Tricot, Guido; Epstein, Joshua

2006-01-01

161

Aseptic technique for cell culture.  

PubMed

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

Coté, R J

2001-05-01

162

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

163

Isolation of (+)-catechin and (-)-epicatechin from Actinidia arguta as bone marrow cell proliferation promoting compounds.  

PubMed

The MeOH extract of stems of Actinidia arguta promoted proliferation of cultured bone marrow cells and stimulated formation of myeloid colonies from bone marrow cells. (+)-Catechin ( 1) and (-)-epicatechin ( 2) were isolated as active compounds from the MeOH extract. Compounds 1 and 2 stimulated the cell proliferation in a concentration-dependent manner in the range of 1 to 100 mg/mL. Compounds 1 and 2 also stimulated formation of myeloid colonies and enhanced the effect of interleukin-3 (IL-3) to increase the number of colony forming-units in culture (CFU-c). In an ex vivo experiment using a model mouse of decreasing bone marrow functions, orally administrated 1 (100 mg/kg/day) stimulated IL-3-induced CFU-c formation of the bone marrow cells. PMID:12709898

Takano, Fumihide; Tanaka, Tomoaki; Tsukamoto, Eiko; Yahagi, Nobuo; Fushiya, Shinji

2003-04-01

164

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

165

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering.  

PubMed

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects. PMID:25241158

Park, Hyun-Ji; Yu, Seung Jung; Yang, Kisuk; Jin, Yoonhee; Cho, Ann-Na; Kim, Jin; Lee, Bora; Yang, Hee Seok; Im, Sung Gap; Cho, Seung-Woo

2014-12-01

166

Principles of cancer cell culture.  

PubMed

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

Cree, Ian A

2011-01-01

167

Observations on the Mechanism of Bone Resorption Induced by Multiple Myeloma Marrow Culture Fluids and Partially Purified Osteoclast-activating Factor  

PubMed Central

Supernatant fluids from the cultures of bone marrow cells from 10 of 12 patients with multiple myeloma (MM) caused bone resorption in organ cultures of fetal rat calvaria. In four patients, the marrow cells were cultured with and without indomethacin (1 ?M). The supernatant fluids from indomethacintreated marrow cultures caused significantly less bone resorption than supernatant fluids of cell cultures without indomethacin. This inhibition of release of bone resorbing factor(s) by myeloma cultures is similar to the previously observed indomethacin-induced inhibition of osteoclast-activating factor (OAF) production by activated human leukocytes. None of the MM supernatants had any effect on cyclic (c)AMP accumulation in resorbing bone in vitro. Four separate preparations of partially purified OAF obtained from phytohemagglutinin-stimulated peripheral human leukocytes were tested for their ability (a) to cause bone resorption in organ cultures of fetal rat and neonatal mouse calvaria and (b) to cause accumulation of cAMP in rat and mouse skeletal tissue in vitro. Those dilutions of OAF that caused bone resorption had no effect on accumulation of cAMP in rat or mouse calvaria incubated in vitro. In addition, no stimulation of adenylate cyclase activity in membranes prepared from fetal rat calvaria could be found. Bone cell populations isolated by sequential collagenase digestion of fetal rat calvaria also showed no cAMP response to these dilutions of OAF. Parathyroid hormone caused a clear response in all three systems. Furthermore, no cAMP response to OAF was observed in calvaria in the presence of cholera toxin (1 ?g/ml) and isobutyl-methylxanthine (0.3 mM). These observations demonstrate that (a) supernatant fluids from MM marrow cultures stimulate bone resorption but do not increase cAMP accumulation in vitro; (b) indomethacin interferes with the release of bone resorbing factors by MM bone marrow cultures suggesting that this process requires prostaglandins; and (c) Sephadex G100 or G75 purified OAF does not stimulate adenylate cyclase or increase cAMP accumulation at equivalent bone resorbing concentrations in rat and mouse skeletal tissue. The resorptive action of MM culture fluids is similar to that of partially purified OAF from activated cultured leukocytes, but different from those of other bone resorbing factors, parathyroid hormone and prostaglandin E2, which stimulate cAMP production in skeletal tissue. PMID:6262378

Josse, Robert G.; Murray, Timothy M.; Mundy, Gregory R.; Jez, Donna; Heersche, Johan N. M.

1981-01-01

168

The Role of Mast Cells in Parathyroid Bone Disease  

PubMed Central

Chronic hyperparathyroidism (HPT) is a common cause of metabolic bone disease. These studies investigated the underlying cellular and molecular mechanisms responsible for the detrimental actions of elevated parathyroid hormone (PTH) on the skeleton. Bone biopsies from hyperparathyroid patients revealed an association between parathyroid bone disease and increased numbers of bone marrow mast cells. We therefore evaluated the role of mast cells in the etiology of parathyroid bone disease in a rat model for chronic HPT. In rats, mature mast cells were preferentially located at sites undergoing bone turnover, and the number of mast cells at the bone–bone marrow interface was greatly increased following treatment with PTH. Time-course studies and studies employing parathyroid hormone–related peptide (PTHrP), as well as inhibitors of platelet-derived growth factor-A (PDGF-A, trapidil), kit (gleevec), and PI3K (wortmannin) signaling revealed that mature mast cell redistribution from bone marrow to bone surfaces precedes and is associated with osteitis fibrosa, a hallmark of parathyroid bone disease. Importantly, mature mast cells were not observed in the bone marrow of mice. Mice, in turn, were resistant to the development of PTH-induced bone marrow fibrosis. These findings suggest that the mast cell may be a novel target for treatment of metabolic bone disease. © 2010 American Society for Bone and Mineral Research. PMID:20200965

Turner, Russell T; Iwaniec, Urszula T; Marley, Kevin; Sibonga, Jean D

2010-01-01

169

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

170

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

171

Cell culture compositions  

DOEpatents

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

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

2014-03-18

172

TOPICAL REVIEW Stem cells in bone tissue engineering  

Microsoft Academic Search

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

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

2010-01-01

173

[The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix].  

PubMed

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration. PMID:16708836

Krugliakov, P V; Sokolova, I B; Zin'kova, N N; Vi?de, S V; Cherednichenko, N N; Kisliakova, T V; Polyntsev, D G

2005-01-01

174

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

175

Silorane resin supports proliferation, differentiation, and mineralization of MLO-A5 bone cells in vitro and bone formation in vivo.  

PubMed

Methyl methacrylate used in bone cements has drawbacks of toxicity, high exotherm, and considerable shrinkage. A new resin, based on silorane/oxirane chemistry, has been shown to have little toxicity, low exotherm, and low shrinkage. We hypothesized that silorane-based resins may also be useful as components of bone cements as well as other bone applications and began testing on bone cell function in vitro and in vivo. MLO-A5, late osteoblast cells, were exposed to polymerized silorane (SilMix) resin (and a standard polymerized bisGMA/TEGDMA methacrylate (BT) resin and compared to culture wells without resins as control. A significant cytotoxic effect was observed with the BT resin resulting in no cell growth, whereas in contrast, SilMix resin had no toxic effects on MLO-A5 cell proliferation, differentiation, nor mineralization. The cells cultured with SilMix produced increasing amounts of alkaline phosphatase (1.8-fold) compared to control cultures. Compared to control cultures, an actual enhancement of mineralization was observed in the silorane resin-containing cultures at days 10 and 11 as determined by von Kossa (1.8-2.0 fold increase) and Alizarin red staining (1.8-fold increase). A normal bone calcium/phosphate atomic ratio was observed by elemental analysis along with normal collagen formation. When used in vivo to stabilize osteotomies, no inflammatory response was observed, and the bone continued to heal. In conclusion, the silorane resin, SilMix, was shown to not only be non cytototoxic, but actually supported bone cell function. Therefore, this resin has significant potential for the development of a nontoxic bone cement or bone stabilizer. PMID:22278990

Eick, J David; Barragan-Adjemian, Cielo; Rosser, Jennifer; Melander, Jennifer R; Dusevich, Vladimir; Weiler, Rachel A; Miller, Bradley D; Kilway, Kathleen V; Dallas, Mark R; Bi, Lianxing; Nalvarte, Elisabet L; Bonewald, Lynda F

2012-04-01

176

Cyclic AMP Signaling Functions as a Bimodal Switch in Sympathoadrenal Cell Development in Cultured Primary Neural Crest Cells  

Microsoft Academic Search

Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development

MATTHEW L. BILODEAU; THERESA BOULINEAU; RONALD L. HULLINGER; OURANIA M. ANDRISANI

2000-01-01

177

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

178

Mesenchymal Stem Cells in Bone Regeneration  

PubMed Central

Significance Mesenchymal stem cells (MSCs) play a key role in fracture repair by differentiating to become bone-forming osteoblasts and cartilage-forming chondrocytes. Cartilage then serves as a template for additional bone formation through the process of endochondral ossification. Recent Advances Endogenous MSCs that contribute to healing are primarily derived from the periosteum, endosteum, and marrow cavity, but also may be contributed from the overlying muscle or through systemic circulation, depending on the type of injury. A variety of growth factor signaling pathways, including BMP, Wnt, and Notch signaling, influence MSC proliferation and differentiation. These MSCs can be therapeutically manipulated to promote differentiation. Furthermore, MSCs can be harvested, cultivated, and delivered to promote bone healing. Critical Issues Pharmacologically manipulating the number and differentiation capacity of endogenous MSCs is one potential therapeutic approach to improve healing; however, ideal agents to influence signaling pathways need to be developed and additional therapeutics that activate endogenous MSCs are needed. Whether isolated and purified, MSCs participate directly in the healing process or serve a bystander effect and indirectly influence healing is not well defined. Future Directions Studies must focus on better understanding the regulation of endogenous MSCs durings fracture healing. This will reveal novel molecules and pathways to therapeutically target. Similarly, while animal models have demonstrated efficacy in the delivery of MSCs to promote healing, more research is needed to understand ideal donor cells, cultivation methods, and delivery before stem cell therapy approaches can be utilized to repair bone. PMID:24527352

Knight, M. Noelle; Hankenson, Kurt D.

2013-01-01

179

Versatile, Fully Automated, Microfluidic Cell Culture System  

E-print Network

Versatile, Fully Automated, Microfluidic Cell Culture System Rafael Go´mez-Sjo1berg, Anne A. Leyrat and quantita- tive cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based

Chen, Christopher S.

180

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

181

Effect of bone graft substitute on marrow stromal cell proliferation and differentiation.  

PubMed

Marrow stromal cells (MSCs) are ideally suited for tissue engineered bone grafts since they have the potential to regenerate bone, but may also maintain the homeostasis of the repaired tissue through their ability for self-renewal. An ideal bone graft substitute should support MSC self-renewal as well as differentiation to ensure complete bone defect regeneration and maintenance. The purpose of this investigation was to determine the effect of different substrate materials on MSC expansion and differentiation. Calcium polyphosphate (CPP), bone and hydroxyapatite/tricalcium phosphate (HA/TCP) were seeded with rat MSCs and maintained in culture conditions that promote cell expansion. At 0, 3, 7, 14, and 21 days cell numbers were determined by measuring their metabolic activity using a MTT assay and the frequency of cycling cells by 24 hr BrdU incorporation. Osteogenic, chondrogenic, and adipogenic marker expression in these cultures was measured by qRT-PCR. An initial drop in cell numbers was observed on all substrates. CPP and bone, but not HA/TCP supported an increase in proliferating cells at day 14 and 21. In addition, no upregulation of mature bone markers was observed in cells cultured on CPP and bone, which suggests that these substrates support the expansion of undifferentiated MSCs. In contrast, cell numbers on HA/TCP decreased with time and only rare BrdU positive cells were observed. This decrease in proliferation correlated with the down regulation of osteogenic progenitor markers and the substantial increase in mature osteocyte markers, indicating that HA/TCP favors MSC differentiation and maturation along the osteogenic lineage. PMID:20336765

Siggers, Kevin; Frei, Hanspeter; Fernlund, Göran; Rossi, Fabio

2010-09-01

182

Giant cell tumour of first metacarpal bone.  

PubMed

Giant cell tumour (GCT) or osteoclastoma is a benign locally aggressive tumour with a tendency for local recurrence. 85-90% of cases occur in long bones; the sites most commonly affected being lower end of femur, upper end of tibia, lower end of radius and proximal humerus in descending order of frequency. Only 2% of GCT occurs in hand. GCT of bone accounts for 5% of all primary bone tumour. 80% of patients are above the age of 18 years, and it occurs commonly in adults between ages of 20 and 40 years. The authors report a case of GCT of first metacarpal which is very rare site for such tumour and only few cases reported in literature so far. PMID:22701064

Shahid, Mohammad; Varshney, Manoranjan; Maheshwari, Veena; Mubeen, Aysha; Gaur, Kavita; Siddiqui, Mohammad

2011-01-01

183

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. PMID:25054153

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

2014-01-01

184

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. PMID:22433781

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

2012-01-01

185

Embryonic stem cells are right now being used around the world to grow different types of cells--heart muscle cells, brain cells, pancreatic cells, liver cells, retinal cells, blood cells, bone cells and much more. Cultures of these cells are  

E-print Network

Embryonic stem cells are right now being used around the world to grow different types of cells and the genetic origins of disease. Embryonic stem cell derived therapies for cell and tissue transplants Administration. Embryonic, adult and other types of stem cell research and clinical uses are all moving ahead due

Wisconsin at Madison, University of

186

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

187

Targeted delivery of mesenchymal stem cells to the bone.  

PubMed

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

Yao, Wei; Lane, Nancy E

2015-01-01

188

Lentiviral tracking of vascular differentiation in bone marrow progenitor cells.  

PubMed

Lentiviral vectors encoding for identifiable marker genes controlled by lineage-specific promoters can be used to track differentiation of bone marrow progenitors into endothelial cells and/or smooth muscle cells. Human VE-Cadherin and Smoothelin-B promoters were cloned into a self-inactivating lentiviral vector (HR-VECad and HR-SMTHB) and used to drive expression of green fluorescent protein (eGFP). These constructs demonstrated specific promoter activity in mature endothelial and smooth muscle cells respectively in vitro. Lin(-) bone marrow progenitor cells (Lin(-) BMCs) in culture were used to test vector ability to track vascular differentiation. HR-VECad transduced Lin(-) BMCs were plated on collagen-coated slides and grown in endothelial media, while HR-SMTHB transduced Lin(-) BMCs were cultured on fibronectin-coated slides and grown in smooth muscle media. For in vivo differentiation assessment, lentiviral transduced Lin(-) BMCs resuspended in Matrigel were injected subcutaneously into C57BL/6J mice. Explants were evaluated for eGFP expression. Lin(-) BMCs grown in endothelial differentiation media produced groups of polygonal endothelial-like cells by days 16-21. When transduced with HR-VECad vector, these expressed eGFP in distinct cells within the colony by days 18-21, and coexpressed VE-Cadherin and eNOS. Lin(-) BMCs grown in smooth muscle differentiation media produced spindle-shaped cells between days 10-14 in culture. When transduced with the HR-SMTHB vector, these cells showed eGFP expression at approximately 12 days, which increased over time and coexpressed alphaSMA, calponin and myosin heavy chain. Within Matrigel plugs containing HR-VECad transduced cells, eGFP(+) constituted 0.4+/-0.2% of total cells. In contrast, within Matrigel plugs containing HR-SMTHB transduced cells, eGFP(+) cells constituted 0.2+/-0.1% of total cells. These data demonstrate the feasibility of selectively marking BMC populations for cell fate determination. PMID:19715661

Schmeckpeper, Jeffrey; Ikeda, Yasuhiro; Kumar, Arun H S; Metharom, Pat; Russell, Stephen J; Caplice, Noel M

2009-01-01

189

Rare giant cell tumor involvement of the olecranon bone  

PubMed Central

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

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

2014-01-01

190

Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissue-engineered bone grafts in critical-size femoral defects  

Microsoft Academic Search

Tissue-engineered bone grafts (TEBG) require highly osteogenic cell sources for use in fracture repair applications. Compared to other sources of mesenchymal stem cells (MSC), human fetal MSC (hfMSC) have recently been shown to be more proliferative and osteogenic. We studied the functional performance of hfMSC-mediated TEBG in 7mm rat femoral critical-sized bone defects (CSD). Dynamically-cultured and osteogenically-primed hfMSC seeded onto

Zhi-Yong Zhang; Swee-Hin Teoh; Mark S. K. Chong; Eddy S. M. Lee; Lay-Geok Tan; Citra N. Mattar; Nicholas M. Fisk; Mahesh Choolani; Jerry Chan

2010-01-01

191

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2010 CFR

...2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2010-01-01

192

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2013 CFR

...2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2013-01-01

193

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2014 CFR

...2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2014-01-01

194

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2012 CFR

...2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2012-01-01

195

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2011 CFR

...2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2011-01-01

196

Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease  

PubMed Central

Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease. PMID:25268740

Garcia-Gomez, Antonio; Las Rivas, Javier De; Ocio, Enrique M.; Díaz-Rodríguez, Elena; Montero, Juan C.; Martín, Montserrat; Blanco, Juan F.; Sanchez-Guijo, Fermín M.; Pandiella, Atanasio; San Miguel, Jesús F.; Garayoa, Mercedes

2014-01-01

197

The role of bone morphogenetic proteins in myeloma cell survival.  

PubMed

Multiple myeloma is characterized by slowly growing clones of malignant plasma cells in the bone marrow. The malignant state is frequently accompanied by osteolytic bone disease due to a disturbed balance between osteoblasts and osteoclasts. Bone morphogenetic proteins (BMPs) are present in the bone marrow and are important for several aspects of myeloma pathogenesis including growth and survival of tumor cells, bone homeostasis, and anemia. Among cancer cells, myeloma cells are particularly sensitive to growth inhibition and apoptosis induced by BMPs and therefore represent good models to study BMP receptor usage and signaling. Our review highlights and discusses the current knowledge on BMP signaling in myeloma. PMID:24853340

Holien, Toril; Sundan, Anders

2014-06-01

198

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

Microsoft Academic Search

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

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

1990-01-01

199

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats  

Microsoft Academic Search

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption\\/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis

F. Boabaid; P. S. Cerri; E. Katchburian

2001-01-01

200

Engraftment of a Clonal Bone Marrow Stromal Cell Line in vivo Stimulates Hematopoietic Recovery from Total Body Irradiation  

Microsoft Academic Search

Whether bone marrow stromal cells of donors contribute physiologically to hematopoietic stem cell reconstitution after marrow transplantation is unknown. To determine the transplantability of nonhematopoietic marrow stromal cells, stable clonal stromal cell line (GB1\\/6) expressing the a isoenzyme of glucose-6-phosphate isomerase (Glu6PI-a, D-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was derived from murine long-term bone marrow cultures and made resistant to neomycin analogue

Pervin Anklesaria; Kenneth Kase; Julie Glowacki; Christie A. Holland; Mary Ann Sakakeeny; Jocyndra A. Wright; T. J. Fitzgerald; Chi-Yu Lee; Joel S. Greenberger

1987-01-01

201

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

202

Crucial factor causing collapse and aggregation of cultured cells in epon resin.  

PubMed

Ultrastructural artifacts regarding collapse and aggregation of cultured cells have been problematic, especially when investigated apoptotic cells. The infiltration process during sample preparation is considered to be the most crucial factor for this problem. This study was conducted using two culture systems: a suspension culture system of human T-lymphocyte Jurkat cells and rabbit mature dendritic cells and a monolayer culture system of human lung macrophages, human breast cancer cells (A-546 cells) and cat bone-invasive gingival cancer cells (sccf3 cells). Fixation was conducted prior to removing or detaching the cells from the culture dishes. Initial infiltration with a 1 : 3 volume ratio of epon resin : propylene oxide was found to be the most crucial step among these cultured cells. The improved epon-resin infiltration method could eliminate the artifacts. Thus, differentiation between artifactual images and true images is highly possible. PMID:25274402

Tangkawattana, Prasarn; Yamaguchi, Mamoru; Klomkleaw, Wuthichai; Niu, Hua; Minaguchi, Jun A; Takehana, Kazushige

2014-01-01

203

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

204

Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics.  

PubMed

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% beta-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications. PMID:16133930

Holtorf, Heidi L; Sheffield, Tiffany L; Ambrose, Catherine G; Jansen, John A; Mikos, Antonios G

2005-09-01

205

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. PMID:23225744

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

206

Effect of chitosan particles and dexamethasone on human bone marrow stromal cell osteogenesis and angiogenic factor secretion  

Microsoft Academic Search

Chitosan is a polysaccharide scaffold used to enhance cartilage repair during treatments involving bone marrow stimulation, and it is reported to increase angiogenesis and osteogenesis in vivo. Here, we tested the hypotheses that addition of chitosan particles to the media of human bone marrow stromal cell (BMSC) cultures stimulates osteogenesis by promoting osteoblastic differentiation and by favoring the release of

Jessica Guzmán-Morales; Hani El-Gabalawy; Minh H. Pham; Nicolas Tran-Khanh; Marc D. McKee; William Wu; Michael Centola; Caroline D. Hoemann

2009-01-01

207

Endothelial cells influence the osteogenic potential of bone marrow stromal cells  

PubMed Central

Background Improved understanding of the interactions between bone cells and endothelial cells involved in osteogenesis should aid the development of new strategies for bone tissue engineering. The aim of the present study was to determine whether direct communication between bone marrow stromal cells (MSC) and human umbilical vein endothelial cells (EC) could influence the osteogenic potential of MSC in osteogenic factor-free medium. Methods After adding EC to MSC in a direct-contact system, cell viability and morphology were investigated with the WST assay and immnostaining. The effects on osteogenic differentiation of adding EC to MSC was systematically tested by the using Superarray assay and results were confirmed with real-time PCR. Results Five days after the addition of EC to MSC in a ratio of 1:5 (EC/MSC) significant increases in cell proliferation and cellular bridges between the two cell types were detected, as well as increased mRNA expression of alkaline phosphatase (ALP). This effect was greater than that seen with addition of osteogenic factors such as dexamethasone, ascorbic acid and ?-glycerophosphate to the culture medium. The expression of transcription factor Runx2 was enhanced in MSC incubated with osteogenic stimulatory medium, but was not influenced by induction with EC. The expression of Collagen type I was not influenced by EC but the cells grown in the osteogenic factor-free medium exhibited higher expression than those cultured with osteogenic stimulatory medium. Conclusion These results show that co-culturing of EC and MSC for 5 days influences osteogenic differentiation of MSC, an effect that might be independent of Runx2, and enhances the production of ALP by MSC. PMID:19919705

2009-01-01

208

Co-culture of bone marrow-derived mesenchymal stem cells overexpressing lipocalin 2 with HK-2 and HEK293 cells protects the kidney cells against cisplatin-induced injury.  

PubMed

Conditioned medium of mesenchymal stem cells (MSCs) is now being used for its cytoprotective effects, especially when the cells are equipped with cytoprotective factors to strengthen them against unfavorable microenvironments. Overexpression of Lcn2 in MSCs mimics in vivo kidney injury. Hence, unraveling how Lcn2-engineered MSCs affect kidney cells has been investigated. Cisplatin treated HK-2 or HEK293 kidney cells were co-cultivated with Lcn2 overexpressing MSCs in upper and lower chambers of transwell plates. Proliferation, apoptosis, and expression of growth factors and cytokines were assessed in the kidney cells. Co-cultivation with the MSCs-Lcn2 not only inhibited cisplatin-induced cytotoxicity in the HK-2 and HEK293 cells, but increased proliferation rate, prevented cisplatin-induced apoptosis, and increased expression of growth factors and the amount of antioxidants in the kidney cells. Thus Lcn2-engineered MSCs can ameliorate and repair injured kidney cells in vitro, which strongly suggests there are beneficial effects of the MSCs-Lcn2 in cell therapy of kidney injury. PMID:25049146

Halabian, Raheleh; Roudkenar, Mehryar Habibi; Jahanian-Najafabadi, Ali; Hosseini, Kamran Mousavi; Tehrani, Hossein Abdul

2015-02-01

209

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

210

“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. PMID:21603148

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

2011-01-01

211

Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone  

PubMed Central

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)–rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into non-myelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis. PMID:21732484

Li, Xin; Ling, Wen; Pennisi, Angela; Wang, Yuping; Khan, Sharmin; Heidaran, Mohammad; Pal, Ajai; Zhang, Xiaokui; He, Shuyang; Zeitlin, Andy; Abbot, Stewart; Faleck, Herbert; Hariri, Robert; Shaughnessy, John D.; van Rhee, Frits; Nair, Bijay; Barlogie, Bart; Epstein, Joshua; Yaccoby, Shmuel

2011-01-01

212

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

213

Growth of human mast cells from bone marrow and peripheral blood-derived CD34(+) pluripotent hematopoietic cells.  

PubMed

Human mast cells (HuMCs) are derived from CD34(+) pluripotent hematopoietic cells which are KIT (CD117)(+) and Fc?RI(-), and lack lineage-specific surface markers. Bone marrow and peripheral blood are the two readily available sources for obtaining CD34(+) cells from which HuMCs can be cultured. CD34(+) cells are isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34(+) cells may be immediately cultured in serum-free culture media containing recombinant human (rh) stem cell factor (SCF), rhIL-6, and rhIL-3 (added only during the first week). Weekly hemidepletions and removal of adherent cells and/or debris enables the investigator to obtain HuMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 weeks. PMID:25388250

Bandara, Geethani; Metcalfe, Dean D; Kirshenbaum, Arnold S

2015-01-01

214

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

215

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

216

Skin-derived precursors differentiate into skeletogenic cell types and contribute to bone repair.  

PubMed

Skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells and that can give rise to peripheral neural and some mesodermal cell types, such as adipocytes. Here, we have asked whether rodent or human SKPs can generate other mesenchymally derived cell types, with a particular focus on osteocytes and chondrocytes. In culture, rodent and human foreskin-derived SKPs differentiated into alkaline-positive, collagen type-1-positive, mineralizing osteocytes, and into collagen type-II-positive chondrocytes that secreted chondrocyte-specific proteoglycans. Clonal analysis demonstrated that SKPs efficiently generated these skeletogenic cell types, and that they were multipotent with regard to the osteogenic and chondrogenic lineages. To ask if SKPs could generate these same lineages in vivo, genetically tagged, undifferentiated rat SKPs were transplanted into a tibial bone fracture model. Over the ensuing 6 weeks, many of the transplanted cells survived within the bone callus, where they were morphologically and phenotypically similar to the endogenous mesenchymal/osteogenic cells. Moreover, some transplanted cells adopted a mature osteocyte phenotype and integrated into the newly formed bone. Some transplanted cells also differentiated into chondrocytes and into smooth muscle cells and/or pericytes that were associated with blood vessels. Thus, both rodent and human SKPs generate skeletogenic cell types in culture, and the injured bone environment is sufficient to instruct SKPs to differentiate down an osteogenic lineage, in a fashion similar to the endogenous mesenchymal precursors. PMID:18834279

Lavoie, Jean-Francois; Biernaskie, Jeffrey A; Chen, Yan; Bagli, Darius; Alman, Benjamin; Kaplan, David R; Miller, Freda D

2009-01-01

217

Osteogenic Effects of Dedifferentiated Fat Cell Transplantation in Rabbit Models of Bone Defect and Ovariectomy-Induced Osteoporosis  

PubMed Central

We have previously reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells have a high proliferative activity and the potential to differentiate into lineages of mesenchymal tissue similar to bone marrow mesenchymal stem cells (MSCs). In the present study, we examined the effects of autologous DFAT cell transplantation on bone regeneration in a rabbit bone defect model and an ovariectomy (OVX)-induced osteoporosis model. The formation of tissue-engineered bone (TEB) was observed when rabbit DFAT cells were loaded onto a ?-tricalcium phosphate (TCP)/collagen sponge and cultured in an osteogenic differentiation medium for 3 weeks. Autologous implantation of DFAT cell-mediated TEB constructs promoted bone regeneration in a rabbit tibial defect model. Regenerated bone tissue induced by transplantation of DFAT cell-mediated TEB constructs was histologically well differentiated and exhibited higher bone strength in a three-point bending test compared to that induced by the ?-TCP/collagen sponge alone. In OVX-induced osteoporosis model rabbits, DFAT cells were obtained with the osteogenic activity similar to cells from healthy rabbits. Intrabone marrow injection of autologous DFAT cells significantly increased the bone mineral density (BMD) at the injected site in the OVX rabbits. Transplanted DFAT cells remained mainly on the injection side of the bone marrow by at least 28 days after intrabone marrow injection and a part of them expressed osteocalcin. In conclusion, these results demonstrate that autologous implantation of DFAT cells contributed to bone regeneration in a rabbit bone defect model and an OVX-induced osteoporosis model. DFAT cells may be an attractive cell source for cell-based bone tissue engineering to treat nonunion fractures in all patients, including those with osteoporosis. PMID:23566022

Kikuta, Shinsuke; Tanaka, Nobuaki; Kazama, Tomohiko; Kazama, Minako; Kano, Koichiro; Ryu, Junnosuke; Tokuhashi, Yasuaki

2013-01-01

218

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

219

Prostate Cancer Cells Promote Osteoblastic Bone Metastases through Wnts  

Microsoft Academic Search

Prostate cancer produces painful osteoblastic bone metasta- ses. Although prostate cancer cells produce numerous osteogenic factors, to date, none have been shown to mediate osteoblastic bone metastases in an in vivo model of prostate cancer. Wnts are a large family of proteins that promote bone growth. Wnt activity is antagonized by endogenous proteins including dickkopf-1 (DKK-1). We explored if prostate

Christopher L. Hall; Anna Bafico; Jinlu Dai; Stuart A. Aaronson; Evan T. Keller

2005-01-01

220

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 vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor ?B ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL:OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior. PMID:22265717

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

2012-06-01

221

Modulation of osteoclastogenesis in porcine bone marrow cultures by quercetin and rutin.  

PubMed

Flavonols, in contrast to soybean isoflavones, are the most abundant phytoestrogens in western diets, being present in onions, beans, fruits, red wine, and tea. They may protect against atherosclerosis, inhibit certain cancer cell types, and reduce bone resorption. The most widely distributed flavonol is quercetin, which occurs mainly as its glycoside, rutin, but data are very scarce regarding the precise mechanism of action of these compounds on bone-resorbing cells at concentrations similar to those detected in human plasma. We have therefore investigated the effects of nanomolar concentrations of quercetin and rutin on the development and activity of osteoclasts in vitro compared with the effects of 17beta-estradiol. Nonadherent porcine bone marrow cells were cultured on dentine slices in the presence of 10 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), with or without 10 nM quercetin, 10 nM rutin or 10 nM 17beta-estradiol for 11 days. Multinuclear TRAP+ cells that resorbed dentine (osteoclasts) developed in the presence of 1,25(OH)2D3, but their number was significantly reduced by quercetin, rutin, and 17beta-estradiol (P < 0.05). Like 17beta-estradiol, both flavonols also significantly reduced resorption (P<0.05) as assessed by the size of pits resorbed on dentine slices. Osteoclasts and osteoclast progenitors contained estrogen receptor alpha (ERalpha), ERbeta, and RANK proteins. Both flavonols increased nuclear ERbeta protein and decreased ERalpha protein of osteoclast progenitors. Moreover, rutin reduced RANK protein, whereas 17beta-oestradiol and quercetin promoted apoptosis by cleavage of caspase-8 and caspase-3. All the effects of flavonols were reversed by 1 microM ICI 182,780, an estrogen antagonist. Thus, the anti-resorbing properties of flavonols are mainly mediated by ER proteins through the inhibition of RANK protein or the activation of caspases. PMID:15688188

Rassi, Claudia M; Lieberherr, Michele; Chaumaz, Gilles; Pointillart, Alain; Cournot, Giulia

2005-03-01

222

Biomimetic bone mechanotransduction modeling in neonatal rat femur organ cultures: structural verification of proof of concept  

PubMed Central

The goal of this work was to develop and validate a whole bone organ culture model to be utilized in biomimetic mechanotransduction research. Femurs harvested from 2-day-old neonatal rat pups were maintained in culture for 1 week post-harvest and assessed for growth and viability. For stimulation studies, femurs were physiologically stimulated for 350 cycles 24 h post-harvest then maintained in culture for 1 week at which time structural tests were conducted. Comparing 1 and 8 days in culture, bones grew significantly in size over the 7-day culture period. In addition, histology supported adequate diffusion and organ viability at 2 weeks in culture. For stimulation studies, 350 cycles of physiologic loading 24 h post-harvest resulted in increased bone strength over the 7-day culture period. In this work, structural proof of concept was established for the use of whole bone organ cultures as mechanotransduction models. Specifically, this work established that these cultures grow and remain viable in culture, are adequately nourished via diffusion and are capable of responding to a brief bout of mechanical stimulation with an increase in strength. PMID:20169394

Saunders, Marnie M.; Simmerman, Linda A.; Reed, Gretchen L.; Sharkey, Neil A.; Taylor, Amanda F.

2010-01-01

223

Bone marrow mesenchymal stem cells are abnormal in multiple myeloma  

Microsoft Academic Search

Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal

J Corre; K Mahtouk; M Attal; M Gadelorge; A Huynh; S Fleury-Cappellesso; C Danho; P Laharrague; B Klein; T Rème; P Bourin

2007-01-01

224

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

Microsoft Academic Search

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

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

2001-01-01

225

Therapy of human tumors in NOD\\/SCID mice with patient-derived reactivated memory T cells from bone marrow  

Microsoft Academic Search

In an analysis of 84 primary-operated breast cancer patients and 11 healthy donors, we found that the bone marrow of most patients contained memory T cells with specificity for tumor-associated antigens. Patients' bone marrow and peripheral blood contained CD8+ T cells that specifically bound HLA\\/peptide tetramers. In short-term culture with autologous dendritic cells pre-pulsed with tumor lysates, patients' memory T

Markus Feuerer; Philipp Beckhove; Lianhua Bai; Erich-Franz Solomayer; Gunther Bastert; Ingo J. Diel; Claudia Pedain; Michael Oberniedermayr; Volker Schirrmacher; Victor Umansky

2001-01-01

226

The effects of canine bone marrow stromal cells on neuritogenesis from dorsal root ganglion neurons in vitro  

Microsoft Academic Search

The present in vitro study was designed to evaluate whether canine bone marrow stromal cells (BMSCs) promote neurite outgrowth from dorsal root\\u000a ganglion (DRG) neurons. Bone marrow aspirates were collected from iliac crests of three young adult dogs. DRG neurons were\\u000a cultured on BMSCs, fibroblasts, or laminin substrates. DRG neurons were also cultured in BMSC- or fibroblast-conditioned media.\\u000a DRG neurons

Hiroaki Kamishina; Jennifer A. Cheeseman; Roger M. Clemmons

2009-01-01

227

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. PMID:23805194

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

228

Generation of clinical grade human bone marrow stromal cells for use in bone regeneration.  

PubMed

In current orthopaedic practice, there is a need to increase the ability to reconstruct large segments of bone lost due to trauma, resection of tumors and skeletal deformities, or when normal regenerative processes have failed such as in non-unions and avascular necrosis. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells), when used in conjunction with appropriate carriers, represent a means by which to achieve bone regeneration in such cases. While much has been done at the bench and in pre-clinical studies, moving towards clinical application requires the generation of clinical grade cells. What is described herein is an FDA-approved cell manufacturing procedure for the ex vivo expansion of high quality, biologically active human BMSCs. This article is part of a Special Issue entitled Stem Cells and Bone. PMID:25064527

Robey, Pamela G; Kuznetsov, Sergei A; Ren, Jiaqiang; Klein, Harvey G; Sabatino, Marianna; Stroncek, David F

2015-01-01

229

Construction of functional tissue-engineered bone using cell sheet technology in a canine model  

PubMed Central

The aim of the present study was to construct functional tissue-engineered bone with cell sheet technology and compare the efficacy of this method with that of traditional bone tissue engineering techniques. Canine bone mesenchymal stem cells (BMSCs) were isolated using density gradient centrifugation and then cultured. The BMSCs were induced to differentiate into osteoblasts and cultured in temperature-responsive culture dishes. The BMSCs detached automatically from the temperature-responsive culture dishes when the temperature was reduced to 20°C, forming an intact cell sheet. Demineralized bone matrix (DBM) and platelet-rich plasma (PRP) were prepared and used to construct a DBM/PRP/BMSC cell sheet/BMSC complex, which was implanted under the left latissimus dorsi muscle in a dog model. A DBM/PRP/BMSC complex was used as a control and implanted under the right latissimus dorsi muscle in the dog model. Immunoblot assays were performed to detect the levels of growth factors. Osteogenesis was observed to be induced significantly more effectively in the DBM/PRP/BMSC cell sheet/BMSC implants than in the DBM/PRP/BMSC implants. Immunoblot assay results indicated that the levels of the growth factors platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) in the experimental group were 3.2- and 2.5-fold higher compared with those in the control group, respectively. These results indicated that the BMSC cell sheets were functional and more effective than the control cell complex. Therefore, cell sheet technology may be used for the effective construction of functional tissue-engineered bone with ideal properties. PMID:24669258

CHEN, TAO; WANG, YANHUI; BU, LINGXUE; LI, NINGYI

2014-01-01

230

Cell Culture as an Alternative in Education.  

ERIC Educational Resources Information Center

Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

Nardone, Roland M.

1990-01-01

231

Cell culture techniques in honey bee research  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

232

Cell Culture Models of Transmissible Spongiform Encephalopathies  

Microsoft Academic Search

In this review, we describe the generation and use of cell culture models of transmissible spongiform encephalopathies, also known as prion diseases. These models include chronically prion-infected cell lines, as well as cultures expressing variable amounts of wild-type, mutated, or chimeric prion proteins. These cell lines have been widely used to investigate the biology of both the normal and the

Florence Béranger; Alain Mangé; Jérôme Solassol; Sylvain Lehmann

2001-01-01

233

In vitro osteogenic induction of bone marrow stromal cells with encapsulated gene-modified bone marrow stromal cells and in vivo implantation for orbital bone repair.  

PubMed

Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12?mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with ?-TCP in vivo. PMID:24498882

Deng, Yuan; Zhou, Huifang; Yan, Chenxi; Wang, Yefei; Xiao, Caiwen; Gu, Ping; Fan, Xianqun

2014-07-01

234

[Polysaccharides of cell cultures of Silene vulgaris].  

PubMed

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

Giunter, E A; Ovodov, Iu S

2007-01-01

235

Cell-Based Approaches to the Engineering of Vascularized Bone Tissue  

PubMed Central

This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo using cell-based therapy approaches. The treatment of large and recalcitrant bone wounds is a serious clinical problem, and in the United States approximately 10% of all fractures are complicated by delayed or non-union. Treatment approaches using growth factor and gene delivery have shown some promise, but results are variable and clinical complications have arisen. Cell-based therapies offer the potential to recapitulate key components of the bone healing cascade, which involves concomitant regeneration of vasculature and new bone tissue. For this reason, osteogenic and vasculogenic cell types have been combined in co-cultures to capitalize on the function of each cell type, and to promote heterotypic interactions. Experiments in both 2D and 3D systems have provided insight into the mechanisms by which osteogenic and vasculogenic cells interact to form vascularized bone, and these approaches have been translated to ectopic and orthotopic models in small animal studies. The knowledge generated by these studies will inform and facilitate the next generation of pre-clinical studies, which are needed to move cell-based orthopaedic repair strategies into the clinic. The science and application of cytotherapy for repair of large and ischemic bone defects is developing rapidly, and promises to provide new treatment methods for these challenging clinical problems. PMID:23999157

Rao, Rameshwar R.; Stegemann, Jan P.

2013-01-01

236

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.  

PubMed

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 ?mol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. PMID:24076187

Ying, Xiaozhou; Sun, Liaojun; Chen, Xiaowei; Xu, Huazi; Guo, Xiaoshan; Chen, Hua; Hong, Jianjun; Cheng, Shaowen; Peng, Lei

2013-12-01

237

J Cell Biochem . Author manuscript Optimizing stem cell culture  

E-print Network

cell culture is widely used in basic research for studying stem cell biology, but also owingJ Cell Biochem . Author manuscript Page /1 7 Optimizing stem cell culture Boudewijn Van Der Sanden * Correspondence should be adressed to: Didier Wion Abstract Stem cells always

Paris-Sud XI, Université de

238

A feeder-free differentiation system identifies autonomously proliferating B cell precursors in human bone marrow.  

PubMed

The peripheral B cell compartment is maintained by homeostatic proliferation and through replenishment by bone marrow precursors. Because hematopoietic stem cells cycle at a slow rate, replenishment must involve replication of precursor B cells. To study proliferation of early human B cell progenitors, we established a feeder cell-free in vitro system allowing the development of B cells from CD34(+) hematopoietic stem cells up to the stage of immature IgM(+) B cells. We found that pro-B and pre-B cells generated in vitro can proliferate autonomously and persist up to 7 wk in culture in the absence of signals induced by exogenously added cytokines. Nevertheless, addition of IL-7 enhanced pre-B cell expansion and inhibited maturation into IgM(+) B cells. The B cell precursor subsets replicating in vitro were highly similar to the bone marrow B cell precursors cycling in vivo. The autonomous proliferation of B cell precursor subsets in vitro and their long-term persistence implies that proliferation during pro-B and pre-B cell stages plays an important role in the homeostasis of the peripheral B cell compartment. Our in vitro culture can be used to study defects in B cell development or in reconstitution of the B cell pool after depletion and chemotherapy. PMID:24379121

Kraus, Helene; Kaiser, Sandra; Aumann, Konrad; Bönelt, Peter; Salzer, Ulrich; Vestweber, Dietmar; Erlacher, Miriam; Kunze, Mirjam; Burger, Meike; Pieper, Kathrin; Sic, Heiko; Rolink, Antonius; Eibel, Hermann; Rizzi, Marta

2014-02-01

239

Response of endothelial cells to decellularized extracellular matrix deposited by bone marrow mesenchymal stem cells  

PubMed Central

Objective: Evaluate the behavior and function of human umbilical vein endothelial cells (HUVECs) on decellularized extracellular matrix (ECM) deposited by bone marrow mesenchymal stem cells (BMSCs). Methods: Prepared through chemical approach, decellularized ECM was characterized by use of immunofluorescence staining. The morphology, attachment, proliferation and migration of HUVECs cultured on six-well tissue culture plastic (TCP) and decellularized ECM were investigated. Results: Decellularized ECM was successfully prepared without three-dimensional architecture disruption. This biological scaffold is similar to nature vascular ECM, preserved various matrix proteins such as type I collagen, type III collagen and fibronection. HUVECs on decellularized ECM showed well attachment and regular arrangement. Decellularized ECM could also significantly enhance the migration and proliferation potential of HUVECs in contrast to TCP. Conclusion: Deposited by BMSCs, ECM can affect the behavior of endothelial cell and could be used as a promising material in tissue engineering.

Xu, Yue; Yan, Mengdie; Gong, Yihong; Chen, Lei; Zhao, Feng; Zhang, Zhaoqiang

2014-01-01

240

Vitamin D metabolism within bone cells: effects on bone structure and strength.  

PubMed

The endocrine activity of 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) contributes to maintaining plasma calcium and phosphate homeostasis through actions on the intestine, kidney and bone. A significant body of evidence has been published over the last 10 years indicating that all major bone cells have the capacity to metabolise 25-hydroxyvitamin D (25(OH)D(3)) to 1,25(OH)(2)D(3), which in turn exerts autocrine/paracrine actions to regulate bone cell proliferation and maturation as well as bone mineralisation and resorption. In vivo and in vitro studies indicate that these autocrine/paracrine activities of 1,25(OH)(2)D(3) in bone tissue contribute to maintaining bone mineral homeostasis and enhancing skeletal health. PMID:21664230

Anderson, Paul H; Atkins, Gerald J; Turner, Andrew G; Kogawa, Masakazu; Findlay, David M; Morris, Howard A

2011-12-01

241

Breast cancer cells induce osteoblast apoptosis: a possible contributor to bone degradation.  

PubMed

Breast cancer cells exhibit a predilection for metastasis to bone. There, the metastases usually bring about bone loss with accompanying pain and loss of function. One way that breast cancer cells disrupt the normal pattern of bone remodeling is by activating osteoclasts, the bone degrading cells. Nevertheless, targeting the osteoclasts does not cure the disease or result in bone repair. These observations indicate that osteoblast function also may be compromised. The objective of this study was to investigate the interaction of metastatic breast cancer cells with osteoblasts. Human metastatic breast cancer cells, MDA-MB-435 or MDA-MB-231, or their conditioned media were co-cultured with a human osteoblast line hFOB1.19. The breast cancer cells caused an increase in the prevalence of apoptotic osteoblasts. Apoptotic osteoblasts detected by the TUNEL assay or by caspase activity increased approximately two to fivefold. This increase was not seen with non-metastatic MDA-MB-468 cells. In an investigation of the mechanism, it was determined that the hFOB1.19 cells expressed fas and that fas was functional. Likewise the hFOB1.19 cells were susceptible to TNF-alpha, but this cytokine was not detected in the conditioned medium of the breast cancer cells. This study indicates that osteoblasts are the target of breast cancer cell-induced apoptosis, but fas/fas-ligand and TNF-alpha, two common initiators of cell death, are probably not involved in this aspect of the metastases/bone cell axis. There are several mechanisms that remain to be explored in order to determine how breast cancer cells bring about osteoblast apoptosis. Even though the specific initiator of apoptosis remains to be identified, the results of this study suggest that the mechanism is likely to be novel. PMID:14743387

Mastro, Andrea M; Gay, Carol V; Welch, Danny R; Donahue, Henry J; Jewell, Jennifer; Mercer, Robyn; DiGirolamo, Douglas; Chislock, Elizabeth M; Guttridge, Kristin

2004-02-01

242

Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells  

PubMed Central

This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high (95 and 85% respectively). Once attached, MCF-7 grow well on both monolayers. Morphology of MCF-7 cells, as analysed by light and epifluorescence microscopy, revealed that MCF-7 cells grow in clusters on 3T3, but disperse on MSC. Concomitant with the lost of their aggregation status, MCF-7 on MSC express low levels of the intercellular adhesion molecules, E-cadherin and epithelial-specific antigen (ESA). These results suggest that MSC represent an appropriate cell target to investigate the cellular and molecular events occurring at the interface of epithelial-marrow stromal interactions. Together, the model here described should permit to further evaluate the significance and prognostic impact of the shift of micrometastatic cells from a cluster-aggregated into a single-cell status. © 2000 Cancer Research Campaign PMID:10755403

Hombauer, H; Minguell, J J

2000-01-01

243

Regeneration of large bone defects in sheep using bone marrow stromal cells.  

PubMed

Bone repair was addressed in a critical-sized defect model in sheep, combining a ceramic biomaterial and mesenchymal progenitor cells. The defects in the tibial mid-diaphysis were treated with autologous bone or with a silicon-stabilized tricalcium phosphate biomaterial, implemented or not by the addition of expanded bone marrow stromal cells. An internal locking compression plate and an external fixator were applied for stabilization. Radiographies were taken during the 8 months follow-up: the pixel grey levels of the lesion areas were determined to evaluate the repair process radiologically. Microradiography, histology and vascular density tests were performed. The autologous bone-treated group performed best, as assessed radiologically, within 20-24 weeks after surgery. Very limited healing was detected in the other experimental group: a partial bone deposition occurred at the periphery of the bony stumps only in the cell-seeded scaffolds. Interestingly, this effect ended within 20-24 weeks, as for the autologous bone, suggesting similar kinetics of the repair processes involved. Moreover, bone deposition was located where a significant reduction of the ceramic scaffold was detected. Faxitron microradiography and histology data confirmed these results. Vascular density analysis evidenced that cell-seeded scaffolds supported an increased vascular ingrowth. Thus, the interactions with the proper microenvironment and the oxygen and nutrient supply in the inner part of the constructs seem fundamental to initiate scaffold substitution and to improve cell performance in tissue-engineered approaches to bone repair. PMID:18537203

Giannoni, P; Mastrogiacomo, M; Alini, M; Pearce, S G; Corsi, A; Santolini, F; Muraglia, A; Bianco, P; Cancedda, R

2008-07-01

244

Culture of Cells from Amphibian Embryos.  

ERIC Educational Resources Information Center

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

Stanisstreet, Martin

1983-01-01

245

Resveratrol increases the bone marrow hematopoietic stem and progenitor cell capacity.  

PubMed

Resveratrol is a plant-derived polyphenol that has shown protective effects against many disorders including, several types of cancers and other age-associated diseases as well as blood disorders in cultured cells and/or animal models. However, whether resveratrol has any impact specifically on normal blood stem cells remains unknown. Here, we show that a 3-week treatment of resveratrol increases the frequency and total numbers of normal bone marrow hematopoietic stem cells (HSC) without any impact on their competitive repopulation capacity. In addition, we show that resveratrol enhances the bone marrow multipotent progenitor capacity in vivo. These results have therapeutic value for disorders of hematopoietic stem and progenitor cells (HSPC) as well as for bone marrow transplantation settings. PMID:25163926

Rimmelé, Pauline; Lofek-Czubek, Sébastien; Ghaffari, Saghi

2014-12-01

246

The utility of human dedifferentiated fat cells in bone tissue engineering in vitro.  

PubMed

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (?-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a ?-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a ?-TCP/CS were well spread and covered the ?-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the ?-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the ?-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an ?-TCP/CS may be an attractive option for bone tissue engineering. PMID:24306271

Sakamoto, Fumito; Hashimoto, Yoshiya; Kishimoto, Naotaka; Honda, Yoshitomo; Matsumoto, Naoyuki

2015-01-01

247

Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models  

NASA Technical Reports Server (NTRS)

An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

Marquette, Michele L.; Sognier, Marguerite A.

2013-01-01

248

Embryonic Stem Cells: Isolation, Characterization and Culture  

NASA Astrophysics Data System (ADS)

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Amit, Michal; Itskovitz-Eldor, Joseph

249

Clonal analysis of bone marrow and macrophage cultures  

SciTech Connect

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

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

1984-01-01

250

AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM  

EPA Science Inventory

Metabolites such as ammonia and lactic formed during mammalian cell culture can frequently be toxic to the cells themselves beyond a threshold concentration of the metabolites. ell culture conducted in the presence of such accumulated metabolites is therefore limited in productiv...

251

Bone marrow Schwann cells induce hematopoietic stem cell hibernation.  

PubMed

Hematopoietic stem cells (HSCs) are clonogenic cells capable of both self-renewal and multilineage differentiation. In adult mouse bone marrow (BM), most HSCs remain in the non-dividing G0-phase of cell cycle, in close contact with supporting cells known as the HSC "niche". In the present study, we focused on signaling mechanisms that regulate stem cell dormancy in the BM niche. We show that TGF-? type II receptor deficiency causes reduced phosphorylation of Smad2/3 and impairs long-term repopulating activity in HSCs, suggesting a significant role for TGF-?/Smad signaling in hematopoiesis. Furthermore, we aimed at defining the candidate BM niche responsible for homeostasis of hematopoiesis, and revealed that non-myelinating Schwann cells sustain HSC hibernation by converting TGF-? from its latent to its active form. PMID:24817152

Yamazaki, Satoshi; Nakauchi, Hiromitsu

2014-06-01

252

Stimulation by bone sialoprotein of calcification in osteoblast-like MC3T3-E1 cells  

Microsoft Academic Search

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the

H.-Y. Zhou; H. Takita; R. Fujisawa; M. Mizuno; Y. Kuboki

1995-01-01

253

Tissue engineering construct on the basis of multipotent stromal adipose tissue cells and Osteomatrix for regeneration of the bone tissue.  

PubMed

We developed a new method of creation of tissue engineering constructs for regeneration of the bone tissue based on the principle of free distribution of cells in a fibrin clot within a scaffold. The tissue engineering construct includes multipotent stromal adipose tissue cells committed in osteogenic lineage, platelet-rich plasma, and resorbed material on the basis of xenogeneic bone collagen. The culture of bone progenitor cells was characterized by the main markers of osteoblastic differon. The material meets all requirements for materials intended for tissue engineering. An innovative high-technological tissue engineering product for clinical application is prepared. PMID:22803063

Bukharova, T B; Arutyunyan, I V; Shustrov, S A; Alekseeva, I S; Fedyunina, I A; Logovskaya, L V; Volkov, A V; Rzhaninova, A A; Grigor'yan, A S; Kulakov, A A; Gol'dshtein, D V

2011-11-01

254

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

PubMed

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

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

2015-02-01

255

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

SciTech Connect

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

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

2010-03-12

256

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

Microsoft Academic Search

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

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

2002-01-01

257

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis  

Microsoft Academic Search

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and

H A Papadaki; M Tsagournisakis; V Mastorodemos; C Pontikoglou; A Damianaki; K Pyrovolaki; K Stamatopoulos; A Fassas; A Plaitakis; G D Eliopoulos

2005-01-01

258

Inhibition of collagen deposition in the extracellular matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures.  

PubMed Central

Long-term production of murine hematopoietic cells in vitro is dependent on establishment of a complex microenvironment consisting of a variety of stromal cells and an extensive extracellular matrix which includes collagen, fibronectin, laminin, proteoglycans, and other undefined components adherent to the culture dishes. Cis-4-hydroxyproline (CHP), a relatively specific inhibitor of collagen secretion, was used to examine the role of extracellular collagen deposition in supporting hematopoiesis in long-term C57B1/6J mouse bone marrow cell cultures. Throughout the 10-wk culture period, all culture dishes contained either 0, 10, 25, or 50 micrograms/ml of CHP. All medium and nonadherent cells were removed at weekly intervals and replaced with fresh medium containing the previous concentrations of CHP. Nonadherent cells were assayed weekly for total cells and pluripotent, erythroid, megakaryocytic, and granulocytic-macrophage progenitor cells. Dishes were killed at selected intervals to assess protein and collagen synthesis in the adherent layer. Adherent cell numbers, as judged by microscopic examination and DNA assays, correlated inversely with CHP concentrations used and paralleled degree of collagen synthesis inhibition. The decreased hemopoietic progenitor cell production correlated closely with percent inhibition of collagen synthesis and stromal cellularity. The CHP concentrations tested were not directly toxic to hemopoietic progenitor cells. These studies demonstrate that collagen deposition in the extracellular matrix of murine bone marrow cell cultures is essential to the establishment of a functional stromal microenvironment that is supportive of long-term hematopoiesis. Images PMID:3980732

Zuckerman, K S; Rhodes, R K; Goodrum, D D; Patel, V R; Sparks, B; Wells, J; Wicha, M S; Mayo, L A

1985-01-01

259

Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy  

PubMed Central

Bone marrow mesenchymal stem cells can differentiate into neurons and astrocytes after transplantation in the spinal cord of rats with ischemia/reperfusion injury. Although bone marrow mesenchymal stem cells are known to protect against spinal cord ischemia/reperfusion injury through anti-apoptotic effects, the precise mechanisms remain unclear. In the present study, bone marrow mesenchymal stem cells were cultured and proliferated, then transplanted into rats with ischemia/reperfusion injury via retro-orbital injection. Immunohistochemistry and immunofluorescence with subsequent quantification revealed that the expression of the axonal regeneration marker, growth associated protein-43, and the neuronal marker, microtubule-associated protein 2, significantly increased in rats with bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Furthermore, the expression of the autophagy marker, microtubule-associated protein light chain 3B, and Beclin 1, was significantly reduced in rats with the bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Western blot analysis showed that the expression of growth associated protein-43 and neurofilament-H increased but light chain 3B and Beclin 1 decreased in rats with the bone marrow mesenchymal stem cell transplantation. Our results therefore suggest that bone marrow mesenchymal stem cell transplantation promotes neurite growth and regeneration and prevents autophagy. These responses may likely be mechanisms underlying the protective effect of bone marrow mesenchymal stem cells against spinal cord ischemia/reperfusion injury. PMID:25374587

Yin, Fei; Meng, Chunyang; Lu, Rifeng; Li, Lei; Zhang, Ying; Chen, Hao; Qin, Yonggang; Guo, Li

2014-01-01

260

Expression of Odontogenic Genes in Human Bone Marrow Mesenchymal Stem Cells  

PubMed Central

Objective: Tooth loss is a common problem and since current tooth replacement methods cannot counter balance with biological tooth structures, regenerating natural tooth structures has become an ideal goal. A challenging problem in tooth regeneration is to find a proper clinically feasible cell to seed.This study was designed to investigate the odontogenic potential of human bone marrow mesenchymal stem cells (HBMSCs) for seeding in tooth regeneration. Materials and Methods: In this experimental study, three pregnant Sprague Dawley (SD) rats were used at the eleventh embryonic day and rat fetuses were removed surgically using semilunar flap under general anesthesia. The primary mandible was cut using a stereomicroscope. The epithelial and mesenchymal components were separated and the dissected oral epithelium was cultured for 3 days. We used flow cytometry analysis to confirm presence of mesenchymal stem cells and not hematopoietic cells and to demonstrate the presence of oral epithelium. Bone marrow mesenchymal stem cells (BMSCs) and cultured oral epithelium were then co-cultured for 14 days. BMSCs cultured alone were used as controls. Expression of two odontogenic genes Pax9 and DMP1 was assessed using quantitative reverse transcription- polymerase chain reaction (RT-PCR). Results: Expression of two odontogenic genes, Pax9 and DMP1, were detected in BMSCs co-cultured with oral epithelium but not in the control group. Conclusion: Expression of Pax9 and DMP1 by human BMSCs in the proximity of odontogenic epithelium indicates odontogenic potential of these cells. PMID:23862115

Mashhadi Abbas, Fatemeh; Sichani Fallahi, Hamed; Khoshzaban, Ahad; Mahdavi, Nazanin; Bagheri, Seyedeh Sara

2013-01-01

261

Studying cell-cell communication in co-culture  

PubMed Central

Heterotypic and homotypic cellular interactions are essential for biological function, and co-culture models are versatile tools for investigating these cellular interactions in vitro. Physiologically relevant co-culture models have been used to elucidate the effects of cell-cell physical contact and/or secreted factors, as well as the influence of substrate geometry and interaction scale on cell response. Identifying the relative contribution of each cell population to co-culture is often experimentally challenging for these cellular interactions studies. In this issue of Biotechnology Journal, Hamilton et al. [1] report on a hydrogel-based co-culture system, that enables paracrine interactions. A simple and elegant method for enzymatic separation of cell populations post co-culture is introduced, thereby enhancing the ease for post-culture analysis of the effects of co-culture on individual cell populations. PMID:23554248

Bogdanowicz, Danielle R.; Lu, Helen H.

2014-01-01

262

REVERSING BONE LOSS BY DIRECTING MESENCHYMAL STEM CELLS TO THE BONE  

PubMed Central

Bone regeneration by systemic transplantation of mesenchymal stem cells (MSCs) is problematic due to the inability to control the MSCs’ commitment, growth and differentiation into functional osteoblasts on the bone surface. Our research group has developed a method to direct the MSCs to the bone surface by conjugating a synthetic peptidomimetic ligand (LLP2A) that has high affinity for activated ?4?1 integrin on the MSC surface, with a bisphosphonates (alendronate) that has high affinity for bone (LLP2A-Ale), to direct the transplanted MSCs to bone. Our in vitro experiments demonstrated that mobilization of LLP2A-Ale to hydroxyapatite accelerated MSC migration that was associated with an increase in the phosphorylation of Akt kinase and osteoblastogenesis. LLP2A-Ale increased the homing of the transplanted MSCs to bone as well as the osteoblast surface, significantly increased the rate of bone formation and restored both trabecular and cortical bone loss induced by estrogen deficiency or advanced age in mice. These results support LLP2A-Ale as a novel therapeutic option to direct the transplanted MSCs to bone for the treatment of established bone loss related to hormone deficiency and aging. PMID:23818248

Yao, Wei; Guan, Min; Jia, Junjing; Dai, Weiwei; Lay, Yu-An E.; Amugongo, Sarah; Liu, Ruiwu; Olivos, David; Saunders, Mary; Lam, Kit; Nolta, Jan; Olvera, Diana; Ritchie, Robert O.; Lane, Nancy E.

2013-01-01

263

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2010 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2010-04-01

264

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2011 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2011-04-01

265

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2013 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2013-04-01

266

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2012 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2012-04-01

267

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.  

Code of Federal Regulations, 2014 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2014-04-01

268

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

PubMed Central

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

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

2014-01-01

269

Characterization of distinct classes of differential gene expression in osteoblast cultures from non-syndromic craniosynostosis bone.  

PubMed

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

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

2014-01-01

270

Effects of Polymer Surfaces on Proliferation and Differentiation of Embryonic Stem Cells and Bone Marrow Stem Cells  

NASA Astrophysics Data System (ADS)

Currently, proliferation and differentiation of stem cell is usually accomplished either in vivo, or on chemical coated tissue culture petri dish with the presence of feeder cells. Here we investigated whether they can be directly cultured on polymeric substrates, in the absence of additional factors. We found that mouse embryonic stem cells did not require gelatin and could remain in the undifferentiated state without feeder cells at least for four passages on partially sulfonated polystyrene. The modulii of cells was measured and found to be higher for cells plated directly on the polymer surface than for those on the same surface covered with gelatin and feeder cells. When plated with feeder cells, the modulii was not sensitive to gelatin. Whereas the differentiation properties of human bone marrow stem cells, which are not adherent, are less dependent on either chemical or mechanical properties of the substrate. However, they behave differently on different toughness hydrogels as oppose to on polymer coated thin films.

Qin, Sisi; Liao, Wenbin; Ma, Yupo; Simon, Marcia; Rafailovich, Miriam

2013-03-01

271

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

PubMed Central

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

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

2014-01-01

272

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

PubMed

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

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

2014-01-01

273

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells.  

PubMed

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(epsilon-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA-PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA-PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA-PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA-PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. PMID:19365613

Hong, Seok-Jung; Jeong, Ishik; Noh, Kyung-Tae; Yu, Hye-Sun; Lee, Gil-Su; Kim, Hae-Won

2009-09-01

274

Bone formation induced by BMP-2 in human osteosarcoma cells  

PubMed Central

Our previous studies demonstrated that BMP-2 inhibits the tumorigenicity of cancer stem cells identified as cells with high aldehyde dehydrogenase activity (ALDH br cells) from the human osteosarcoma cell line OS99-1. We further investigated whether BMP-2 is capable of inducing bone formation in OS99-1 cells. Flow cytometry sorting was used to isolate tumorigenic ALDH br and non-tumorigenic ALDH lo cells. qRT-PCR was used to quantify the gene expression. A xenograft model was used to verify the bone formation in vivo . There was significantly higher mRNA expression of BMPR1B and BMPR2 in ALDH lo cells compared with that in ALDH br cells and the BMPR1B expression in ALDH lo cells was ?8-fold higher compared to that in ALDH br cells. BMP-2 was also found to induce higher transcription of osteogenic markers Runx-2, Osterix (Osx), alkaline phosphatase (ALP) and collagen type I in ALDH lo cells compared to ALDH br cells, which were mediated by the canonical Smad signaling pathway. In vivo , BMP-2 was identified to induce bone formation in both ALDH br and ALDH lo cells. All animals receiving 1×10 4 ALDH lo cells treated with 30 ? g of BMP-2 per animal showed bone formation within 1–2 weeks after injection in mice. Bone formation induced by BMP-2 in ALDH lo cells showed significantly more bone mineral content compared to that in ALDH br cells. BMP-2 induces bone formation in heterogeneous osteosarcoma cells and BMP-2 may have a promising therapeutic role for treating human osteosarcoma by inducing differentiation along an osteogenic pathway. PMID:23900689

WANG, LIN; PARK, PAUL; LA MARCA, FRANK; THAN, KHOI; RAHMAN, SHAYAN; LIN, CHIA-YING

275

Bone formation induced by BMP-2 in human osteosarcoma cells.  

PubMed

Our previous studies demonstrated that BMP-2 inhibits the tumorigenicity of cancer stem cells identified as cells with high aldehyde dehydrogenase activity (ALDH(br) cells) from the human osteosarcoma cell line OS99-1. We further investigated whether BMP-2 is capable of inducing bone formation in OS99-1 cells. Flow cytometry sorting was used to isolate tumorigenic ALDH(br) and non-tumorigenic ALDH(lo) cells. qRT-PCR was used to quantify the gene expression. A xenograft model was used to verify the bone formation in vivo. There was significantly higher mRNA expression of BMPR1B and BMPR2 in ALDH(lo) cells compared with that in ALDH(br) cells and the BMPR1B expression in ALDH(lo) cells was ~8-fold higher compared to that in ALDHbr cells. BMP-2 was also found to induce higher transcription of osteogenic markers Runx-2, Osterix (Osx), alkaline phosphatase (ALP) and collagen type I in ALDH(lo) cells compared to ALDH(br) cells, which were mediated by the canonical Smad signaling pathway. In vivo, BMP-2 was identified to induce bone formation in both ALDH(br) and ALDH(lo) cells. All animals receiving 1 x 10()4 ALDH(lo) cells treated with 30 µg of BMP-2 per animal showed bone formation within 1-2 weeks after injection in mice. Bone formation induced by BMP-2 in ALDH(lo) cells showed significantly more bone mineral content compared to that in ALDH(br) cells. BMP-2 induces bone formation in heterogeneous osteosarcoma cells and BMP-2 may have a promising therapeutic role for treating human osteosarcoma by inducing differentiation along an osteogenic pathway. PMID:23900689

Wang, Lin; Park, Paul; La Marca, Frank; Than, Khoi; Rahman, Shayan; Lin, Chia-Ying

2013-10-01

276

Stem cells in skeletal physiology and endocrine diseases of bone.  

PubMed

Self-renewing, multipotent progenitors of skeletal tissues are found within skeletal segments (skeletal stem cells) and coincide with adventitial reticular cells of bone marrow vessels in situ and with explanted clonogenic stromal cells ex vivo. These cells, which can be identified and prospectively isolated based on a minimal surface phenotype noted for expression of CD146, CD105 and alkaline phosphatase, are established during bone development through interactions with developing sinusoids. They represent a crucial crossroad of skeletal and hematopoietic physiology, as well as of endocrine regulation of bone growth and remodeling. In addition, they are central to major endocrine functions of bone itself, such as regulation of renal phosphate handling. Skeletal stem cells represent a central model system for investigating skeletal diseases, as tools for in vitro and in vivo models, for cell therapy-based strategies, or as targets for drugs. PMID:21865758

Bianco, Paolo; Sacchetti, Benedetto; Riminucci, Mara

2011-01-01

277

Constructing a 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)

1996-01-01

278

Comparison of osteogenic ability of rat mesenchymal stem cells from bone marrow, periosteum, and adipose tissue.  

PubMed

Mesenchymal stem cells (MSCs) reside in many types of tissue and are able to differentiate into various functional cells including osteoblasts. Recently, adipose tissue-derived MSCs (AMSCs) have been shown to differentiate into many lineages, and they are considered a source for tissue regeneration. The purpose of this study was to compare the osteogenic differentiation capability of MSCs from bone marrow (BMSCs), MSCs from periosteum (PMSCs), and AMSCs using in vitro culture and in vivo implantation experiments. We harvested these MSCs from 7-week-old rats. The cells were seeded and cultured for 7 days in primary culture to assay a colony-forming unit. The frequency of the unit was the smallest in the BMSCs (P < 0.001). After primary culture, subculture was performed under osteogenic differentiation conditions for 1 and 2 weeks to detect mineralization as well as the bone-specific proteins of alkaline phosphatase and osteocalcin as osteogenic markers. BMSCs and PMSCs showed distinct osteogenic differentiation capability in comparison with other MSCs (P < 0.001). For the in vivo assay, composites of these cells and hydroxyapatite ceramics were subcutaneously implanted into syngeneic rats and harvested after 6 weeks. Micro-computed tomographic (CT) and histological analyses demonstrated that new bone formation was detected in the composites using BMSCs and PMSCs, although it was hard to detect in other composites. The CT analyses also demonstrated that the bone volume of BMSC composites was more than that of AMSC composites (P < 0.001). These results indicate that BMSCs and PMSCs could be ideal candidates for utilization in practical bone tissue regeneration. PMID:18305886

Hayashi, Ousuke; Katsube, Yoshihiro; Hirose, Motohiro; Ohgushi, Hajime; Ito, Hiromoto

2008-03-01

279

Emulsions Containing Perfluorocarbon Support Cell Cultures  

NASA Technical Reports Server (NTRS)

Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.

1990-01-01

280

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

E-print Network

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

Abraham, Nader G.

281

Cannabinoids induce incomplete maturation of cultured human leukemia cells  

SciTech Connect

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

1987-08-01

282

The Impact of Simulated and Real Microgravity on Bone Cells and Mesenchymal Stem Cells  

PubMed Central

How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering. PMID:25110709

Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Magnusson, Nils; Infanger, Manfred; Grosse, Jirka; Eilles, Christoph

2014-01-01

283

Connective Tissue Growth Factor reporter mice label a subpopulation of mesenchymal progenitor cells that reside in the trabecular bone region.  

PubMed

Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously had been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed that it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. PMID:25464947

Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter

2015-02-01

284

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2013-04-01

285

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2012-04-01

286

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2011-04-01

287

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2010-04-01

288

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2014 CFR

...2014-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2014-04-01

289

Effect of coating Straumann Bone Ceramic with Emdogain on mesenchymal stromal cell hard tissue formation.  

PubMed

Periodontal tissue engineering requires a suitable biocompatible scaffold, cells with regenerative capacity, and instructional molecules. In this study, we investigated the capacity of Straumann Bone Ceramic coated with Straumann Emdogain, a clinical preparation of enamel matrix protein (EMP), to aid in hard tissue formation by post-natal mesenchymal stromal cells (MSCs) including bone marrow stromal cells (BMSCs) and periodontal ligament fibroblasts (PDLFs). MSCs were isolated and ex vivo-expanded from human bone marrow and periodontal ligament and, in culture, allowed to attach to Bone Ceramic in the presence or absence of Emdogain. Gene expression of bone-related proteins was investigated by real time RT-PCR for 72 h, and ectopic bone formation was assessed histologically in subcutaneous implants of Bone Ceramic containing MSCs with or without Emdogain in NOD/SCID mice. Alkaline phosphatase activity was also assessed in vitro, in the presence or absence of Emdogain. Collagen-I mRNA was up-regulated in both MSC populations over the 72-h time course with Emdogain. Expression of BMP-2 and the osteogenic transcription factor Cbfa-1 showed early stimulation in both MSC types after 24 h. In contrast, expression of BMP-4 was consistently down-regulated in both MSC types with Emdogain. Up-regulation of osteopontin and periostin mRNA was restricted to BMSCs, while higher levels of bone sialoprotein-II were observed in PDLFs with Emdogain. Furthermore, alkaline phosphatase activity levels were reduced in both BMSCs and PDLFs in the presence of Emdogain. Very little evidence was found for ectopic bone formation following subcutaneous implantation of MSCs with Emdogain-coated or -uncoated Bone Ceramic in NOD/SCID mice. The early up-regulation of several important bone-related genes suggests that Emdogain may have a significant stimulatory effect in the commitment of mesenchymal cells to osteogenic differentiation in vitro. While Emdogain inhibited AP activity and appeared not to induce ectopic bone formation, longer-term studies are required to determine whether it promotes the final stages of osteoblast formation and mineralization at gene and protein levels. While used in clinical applications, whether Emdogain and other commercial preparations of EMPs truly possess the capacity to induce the regeneration of bone or other components of the periodontium remains to be established. PMID:21584694

Mrozik, Krzysztof Marek; Gronthos, Stan; Menicanin, Danijela; Marino, Victor; Bartold, P Mark

2012-06-01

290

Augmentation of bone healing of nonunion fracture using stem cell based tissue engineering in a dog: a case report  

Microsoft Academic Search

A 4-year-old, intact male crossbreed dog, weighing 27 kg, was referred for the treatment of a nonun- ion fracture. The radiographs revealed displacement of the radius and ulna bone fracture fragment and a sclerotic fracture end of the radius. Autologous adipose derived stem cells (ADSCs) were isolated and expanded ex vivo in a culture. The ADSCs (3.2 × 107 cells)

H. B. Lee; Y. S. Chung; S. Y. Heo; N. S. Kim

291

The Yield of Bone Marrow Biopsy and Culture Compared with Blood Culture in the Evaluation of HIV-infected Patients for Mycobacterial and Fungal Infections  

Microsoft Academic Search

Purpose: To compare the clinical utility of bone marrow biopsy and culture specimens with blood cultures for mycobacterial and fungal infections among human immunodeficiency virus (HIV)-infected patients.Patients and Methods: All bone marrow biopsies obtained from HIV-infected patients at the University of Alabama at Birmingham (UAB) Medical Center during 1993 to 1995 were blindly reviewed in a standardized format. Bone marrow

J. Michael Kilby; Marisa B Marques; David L Jaye; Paul B Tabereaux; Vishnu B Reddy; Ken B Waites

1998-01-01

292

Multiple melanocortin receptors are expressed in bone cells  

NASA Technical Reports Server (NTRS)

Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

2005-01-01

293

Chondrogenesis of mesenchymal stem cells in an osteochondral environment is mediated by the subchondral bone.  

PubMed

In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGF?) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGF?; however, the observed results could not be attributed to TGF?, as either capturing TGF? with an antibody or blocking the canonical TGF? signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGF? family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role. PMID:23980750

de Vries-van Melle, Marloes L; Narcisi, Roberto; Kops, Nicole; Koevoet, Wendy J L M; Bos, P Koen; Murphy, J Mary; Verhaar, Jan A N; van der Kraan, Peter M; van Osch, Gerjo J V M

2014-01-01

294

Different titanium surfaces modulate the bone phenotype of SaOS-2 osteoblast-like cells.  

PubMed

Commercially pure titanium implants presenting a relatively smooth, machined surface or a roughened endosseous surface show a large percentage of clinical success. Surface properties of dental implants seem to affect bone cells response. Implant topography appears to modulate cell growth and differentiation of osteoblasts affecting the bone healing around the titanium implant. The aim of the present study was to examine the effects of 1cm diameter and 1mm thick titanium disks on cellular morphology, adhesion and bone phenotypic expression of human osteoblast-like cells, SaOS-2. SaOS-2 cells were cultured on commercially 1 cm pure titanium disks with three different surface roughness: smooth (S), sandblasted (SB) and titanium plasma sprayed (TPS). Differences in the cellular morphology were found when they were grown on the three different surfaces. An uniform monolayer of cells recovered the S surface, while clusters of multilayered irregularly shaped cells were distributed on the rough SB and TPS surfaces. The adhesion of SaOS-2 cells, as measured after 3h of culture, was not affected by surface roughness. ECM components such as Collagen I (CoI), Fibronectin (FN), Vitronectin (VN) and Tenascin (TN) were secreted and organized only on the SB and TPS surfaces while they remained into the cytoplasm on the S surfaces. Osteopontin and BSP-II were largely detected on the SB and TPS surfaces, while only minimal production was observed on the S ones. These data show that titanium surface roughness affects bone differentiation of osteoblast like-cells, SaOS-2, indicating that surface properties may be able to modulate the osteoblast phenotype. These observations also suggest that the bone healing response around dental implants can be affected by surface topography. PMID:15590412

Postiglione, L; Di Domenico, G; Ramaglia, L; di Lauro, A E; Di Meglio, F; Montagnani, S

2004-01-01

295

Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells.  

PubMed

Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as "cell factories" that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies. PMID:25411477

Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon; Van Vliet, Krystyn J

2015-01-01

296

Bone-marrow haematopoietic-stem-cell niches  

Microsoft Academic Search

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

Anne Wilson; Andreas Trumpp

2006-01-01

297

Cell culture processes for monoclonal antibody production  

PubMed Central

Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

2010-01-01

298

Implant surface coatings with bone sialoprotein, collagen, and fibronectin and their effects on cells derived from human maxillar bone.  

PubMed

The interaction between implant material and surrounding tissues is believed to play a fundamental role in implant success. Although bone sialoprotein (BSP) has been found to be osteoinductive when coated onto femoral implants, collagen and fibronectin are the most used compounds for preparation of pre-coated cell culture slides at present. In this study, the support of BSP-, collagen- and fibronectin-coated and non-coated implant material for the development of adult human maxillar bone in vitro was studied and compared. The expression of bone turnover markers like BSP and osteocalcin as well as osteonectin, transforming growth factor beta (TGF-beta) and CD90 during different time periods of cell cultivation (3, 5, 10, 15, 20 and 25 days) was visualized immunohistochemically. The distribution patterns of the cells were examined on a rough surface of the titanium-hydroxyapatite dental implant material TICER and on a total smooth surface of the technical implant material glimmer. Significantly different values were found for glimmer at the 15. and the 20. Div, exclusively, indicating that a smooth surface was more improved than a rough ceramic surface by pre-coatings. The White-test using rankings of the median values gave evidence for BSP-coatings at position 1 followed by collagen. Our experiments were designed to use very low concentrated BSP coating solution with the aim to reduce the healing time with a minimal effort and minimal risks for the patients. PMID:17363352

Hilbig, H; Kirsten, M; Rupietta, R; Graf, H-L; Thalhammer, S; Strasser, S; Armbruster, F P

2007-01-31

299

Advances in cell culture: anchorage dependence.  

PubMed

Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000-6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems--microcarrier/microcarrier-clump cultures using stirred-tank reactors--for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

Merten, Otto-Wilhelm

2015-02-01

300

Cultured Porcine Coronary Artery Smooth Muscle Cells  

Microsoft Academic Search

Abstract—Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage

Thomas Christen; Marie-Luce Bochaton-Piallat; Pascal Neuville; Sander Rensen; Mireille Redard; Guillaume van Eys; Giulio Gabbiani

301

Gene expression profile of human bone marrow stromal cells determined by restriction fragment differential display analysis.  

PubMed

Using an in vitro osteogenic culture system, we carried out a restriction fragment differential display (RFDD-PCR) to identify genes expressed by these cells in their undifferentiated stage and not expressed, or expressed at a lower level, in a closely related but distinct cell type: bone marrow stromal cells (BMSC)-derived osteoblasts (BDO). Forty-seven candidate regulated genes, selected by RFDD, were analyzed by RT-PCR analysis in three cell clones and in primary cultures from seven different donors. A subset of three genes were confirmed as upregulated in BMSC relative to BDO in every primary culture and cloned population examined: betaIG-h3, IGFbp3, and LOXL2. Their differential expression was confirmed by Northern analysis and the corresponding proteins were detected by immunolocalization in BMSC. PMID:15211571

Monticone, Massimiliano; Liu, Yi; Tonachini, Laura; Mastrogiacomo, Maddalena; Parodi, Stefano; Quarto, Rodolfo; Cancedda, Ranieri; Castagnola, Patrizio

2004-07-01

302

Identification and Purification of Mesodermal Progenitor Cells From Human Adult Bone Marrow  

PubMed Central

Bone marrow–derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine. PMID:18991503

Pacini, Simone; Trombi, Luisa; Fazzi, Rita; Montali, Marina; Ikehara, Susumu; Abraham, Nader G.

2009-01-01

303

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

PubMed

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

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

2015-01-01

304

Bone marrow stromal cell-derived extracellular matrix promotes osteogenesis of adipose-derived stem cells.  

PubMed

Adipose-derived stem cells (ASCs) can differentiate into multiple cell lineages and favor adipogenesis rather than osteogenesis. Because the extracellular matrix (ECM) component of the stem cell niche is important in stem cell differentiation, we hypothesized that ECM produced by human bone marrow stromal cells (BM-ECM) could enhance the osteogenic potential of ASCs during in vitro expansion. We have compared the replication and osteogenic differentiation of ASCs expanded on BM-ECM versus tissue culture plastic (TCP) in vitro and in vivo. During the first two passages, ASC proliferation on BM-ECM was 3.27-fold greater than that on TCP. ASCs expanded on BM-ECM formed more osteogenic colonies and higher expression of osteogenic markers than ASCs expanded on TCP. In nude mice, ASCs that had been expanded on BM-ECM formed more new bone tissue than those expanded on TCP. The data indicate that BM-ECM can be used to promote the osteogenic fate of ASCs. PMID:25264269

Zhang, Zhiliang; Luo, Xuan; Xu, Haisong; Wang, Lin; Jin, Xin; Chen, Rui; Ren, Xiaoyun; Lu, Yi; Fu, Mingang; Huang, Yixiong; He, Jiyin; Fan, Zhihong

2015-03-01

305

Dimethyloxaloylglycine Improves Angiogenic Activity of Bone Marrow Stromal Cells in the Tissue-Engineered Bone  

PubMed Central

One of the big challenges in tissue engineering for treating large bone defects is to promote the angiogenesis of the tissue-engineered bone. Hypoxia inducible factor-1? (HIF-1?) plays an important role in angiogenesis-osteogenesis coupling during bone regeneration, and can activate a broad array of angiogenic factors. Dimethyloxaloylglycine (DMOG) can activate HIF-1? expression in cells at normal oxygen tension. In this study, we explored the effect of DMOG on the angiogenic activity of bone mesenchymal stem cells (BMSCs) in the tissue-engineered bone. The effect of different concentrations of DMOG on HIF-1a expression in BMSCs was detected with western blotting, and the mRNA expression and secretion of related angiogenic factors in DMOG-treated BMSCs were respectively analyzed using qRT-PCR and enzyme linked immunosorbent assay. The tissue-engineered bone constructed with ?-tricalcium phosphate (?-TCP) and DMOG-treated BMSCs were implanted into the critical-sized calvarial defects to test the effectiveness of DMOG in improving the angiogenic activity of BMSCs in the tissue-engineered bone. The results showed DMOG significantly enhanced the mRNA expression and secretion of related angiogenic factors in BMSCs by activating the expression of HIF-1?. More newly formed blood vessels were observed in the group treated with ?-TCP and DMOG-treated BMSCs than in other groups. And there were also more bone regeneration in the group treated with ?-TCP and DMOG-treated BMSCs. Therefore, we believed DMOG could enhance the angiogenic activity of BMSCs by activating the expression of HIF-1?, thereby improve the angiogenesis of the tissue-engineered bone and its bone healing capacity. PMID:25013382

Ding, Hao; Chen, Song; Song, Wen-Qi; Gao, You-Shui; Guan, Jun-Jie; Wang, Yang; Sun, Yuan; Zhang, Chang-Qing

2014-01-01

306

Isolation of mitochondria from tissue culture cells.  

PubMed

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ?10? cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation. PMID:25275104

Clayton, David A; Shadel, Gerald S

2014-10-01

307

Culture and Manipulation of Embryonic Cells  

PubMed Central

The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

Edgar, Lois G.; Goldstein, Bob

2012-01-01

308

Cell Culture on MEMS Platforms: A Review  

E-print Network

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods ...

Ni, Ming

309

Effects of Electromagnetic Stimuli on Bone and Bone Cells in vitro: Inhibition of Responses to Parathyroid Hormone by Low-Energy Low-Frequency Fields  

Microsoft Academic Search

Low-energy electromagnetic fields pulsed at frequencies of 10-90 Hz significantly increase healing of chronic fracture nonunions in man. These fields are effective at tissue current levels several orders of magnitude lower than those required for transmembrane depolarization of normal cells. We have examined the effects of two clinically used pulsed electromagnetic fields on cultures of the osteoblast-like mouse bone cell

Richard A. Luben; Christopher D. Cain; Monica Chi-Yun Chen; David M. Rosen; W. Ross Adey

1982-01-01

310

Magnesium variability of lymphocytes from cell culture.  

PubMed

We determined the magnesium content for two different Burkitt's lymphoma cell lines (EW 36 and CA 46) after transfer to new media for 7 consecutive days. Aliquots of the cell culture were washed and the cell pellet was obtained by centrifugation and lysed with distilled water with and without the addition of 0.25% lanthanum oxide. Magnesium was determined by atomic absorption spectrophotometry. The addition of lanthanum permitted the detection of between 8 and 40% more magnesium. The increased magnesium liberated by the addition of lanthanum was calculated as the "bound" magnesium. The results show that the total magnesium is inversely related and the bound magnesium directly related to the age of the cell culture. Thus, there is a decrease of lymphocyte magnesium content and an increase in the percentage of magnesium bound for these two cell lines with increasing age of the cell culture. PMID:4078199

Hosseini, J M; Elin, R J

1985-01-01

311

Effects of Spaceflight on Cells of Bone Marrow Origin  

PubMed Central

Once only a subject for science fiction novels, plans for establishing habitation on space stations, the Moon, and distant planets now appear among the short-term goals of space agencies. This article reviews studies that present biomedical issues that appear to challenge humankind for long-term spaceflights. With particularly focus on cells of bone marrow origin, studies involving changes in bone, immune, and red blood cell populations and their functions due to extended weightlessness were reviewed. Furthermore, effects of mechanical disuse on primitive stem cells that reside in the bone marrow were also included in this review. Novel biomedical solutions using space biotechnology will be required in order to achieve the goal of space exploration without compromising the functions of bone marrow, as spaceflight appears to disrupt homeostasis for all given cell types. Conflict of interest:None declared. PMID:24385745

Özçivici, Engin

2013-01-01

312

Human Bone-Forming Chondrocytes Cultured in the Hydrodynamic Focusing Bioreactor Retain Matrix Proteins: Similarities to Spaceflight Results  

NASA Technical Reports Server (NTRS)

Fracture healing, crucial to a successful Mars mission, involves formation of a cartilaginous fracture callus which differentiates, mineralizes, ossifies and remodels via the endochondral process. Studies of spaceflown and tailsuspended rats found that, without loading, fracture callus formation and cartilage differentiation within the callus were minimal. We found delayed differentiation of chondrocytes within the rat growth plate on Cosmos 1887, 2044, and Spacelab 3. In the current study, differentiation of human bone-forming chondrocytes cultured in the hydrodynamic focusing bioreactor (HFB) was assessed. Human costochondral chondrocytes in suspension were aggregated overnight, then cultured in the HFB for 25 days. Collagen Type II, aggrecan and unsulfated chondroitin were found extracellularly and chondroitin sulfates 4 and 6 within the cell. Lack of secretion was also found in pancreatic cells of spaceflown rats, and in our SL3 studies. The HFB can be used to study cartilage differentiation in simulated microgravity.

Duke, P. J.; Hecht, J.; Montufar-Solis, D.

2006-01-01

313

Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.  

PubMed

In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPAR?2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPAR?2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration. PMID:24573839

Tansriratanawong, Kallapat; Tamaki, Yuichi; Ishikawa, Hiroshi; Sato, Soh

2014-10-01

314

In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow.  

PubMed

Mesenchymal stem cells derived from human teeth and bone marrow have been characterized by many research groups, but demonstrate inconsistent cellular phenotypes or functions, partly because of differences in culture methodology. Therefore, our aims were to resolve these inconsistencies and discuss the potential uses of these cells in research/clinical applications. We isolated and characterized dental stem cells (DSCs) from the dental pulp, periodontal ligament, apical papilla (APSCs) and dental follicle (DFSCs) of mature and immature teeth, along with bone marrow-derived stem cells (BMSCs) from the iliac crest. We compared the clonogenic and proliferative potentials of these cells in terms of colony-forming efficiency, proliferation potential, population doubling time and cell cycle. All DSCs, particularly APSCs and DFSCs, possessed greater proliferative potential than BMSCs. All stem cells expressed typical mesenchymal and embryonic markers, and developed alizarin red-positive mineralization nodules and Oil red O-positive lipid droplets when cultured in osteogenic and adipogenic media, respectively. Immunocytochemistry revealed that all stem cells developed neuronal markers when cultured in a control medium without neural inductive supplements. After 7 days of neurogenic culture, the differentiated cells showed a transition from fibroblast-like to neuron-like cell bodies with long processes, suggesting that the stem cells differentiated into mature neurons. Karyotyping confirmed that the stem cells maintained a normal karyotype and were chromosomally stable. Our results provide new insights into the physiological properties of stem cells with a normal karyotype and indicate that DSCs are appropriate for basic research and clinical applications. PMID:22772774

Tamaki, Yuichi; Nakahara, Taka; Ishikawa, Hiroshi; Sato, Soh

2013-07-01

315

High-Frequency Vibration Treatment of Human Bone Marrow Stromal Cells Increases Differentiation toward Bone Tissue  

PubMed Central

In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30?Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient. PMID:23585968

Prè, D.; Ceccarelli, G.; Visai, L.; Benedetti, L.; Imbriani, M.; Cusella De Angelis, M. G.; Magenes, G.

2013-01-01

316

The use of rat, rabbit or human bone marrow derived cells for cytocompatibility evaluation of metallic elements.  

PubMed

Rat, rabbit and human bone marrow cells were cultured according to the method previously reported for cells of rat origin [1] and were exposed, or not (control), to corrosion products of a Co-Cr orthopaedic alloy as well as to metal salts containing Co2+, Cr3+ and Cr6+. Cells were cultured for 21 days and analysed for the following biochemical parameters: intracellular MTT reduction (i.e. cell viability/proliferation), alkaline phosphatase (ALP) activity and protein production. Morphological observations included both histochemistry (detection of ALP-positive cells, calcium and phosphate deposits) and scanning electron microscopy (SEM). Control cultures of rat and rabbit cells showed higher proliferation rates than human cells at the start of culture, but they all reached similar values on day 21. Protein production was parallel to cell proliferation. In contrast, ALP activity of rat cultures was much stronger than rabbit or human cultures. All cell types were able to develop the osteogenic phenotype in vitro.Co-Cr extract caused inhibitory effects on cell viability, on ALP activity and, to a lower extent, on protein production of all rat, rabbit and human cell cultures. Compared to rat and rabbit cultures, human cultures were the most sensitive to metal ions exposure. PMID:15348764

Tomás, H; Carvalho, G S; Fernandes, M H; Freire, A P; Abrantes, L M

1997-04-01

317

Biomaterials and bone mechanotransduction  

NASA Technical Reports Server (NTRS)

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

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

2001-01-01

318

Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells  

PubMed Central

Background Bone marrow-derived mesenchymal stem cells (BMSCs) are a widely researched adult stem cell population capable of differentiation into various lineages. Because many promising applications of tissue engineering require cell expansion following harvest and involve the treatment of diseases and conditions found in an aging population, the effect of donor age and ex vivo handling must be understood in order to develop clinical techniques and therapeutics based on these cells. Furthermore, there currently exists little understanding as to how these two factors may be influenced by one another. Results Differences in the adipogenic, chondrogenic, and osteogenic differentiation capacity of murine MSCs harvested from donor animals of different age and number of passages of these cells were observed. Cells from younger donors adhered to tissue culture polystyrene better and proliferated in greater number than those from older animals. Chondrogenic and osteogenic potential decreased with age for each group, and adipogenic differentiation decreased only in cells from the oldest donors. Significant decreases in differentiation potentials due to passage were observed as well for osteogenesis of BMSCs from the youngest donors and chondrogenesis of the cells from the oldest donors. Conclusion Both increasing age and the number of passages have lineage dependent effects on BMSC differentiation potential. Furthermore, there is an obvious interplay between donor age and cell passage that in the future must be accounted for when developing cell-based therapies for clinical use. PMID:18957087

Kretlow, James D; Jin, Yu-Qing; Liu, Wei; Zhang, Wen Jie; Hong, Tan-Hui; Zhou, Guangdong; Baggett, L Scott; Mikos, Antonios G; Cao, Yilin

2008-01-01

319

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart.  

PubMed

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit(+) CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit(+) CSCs. PMID:19912439

Barile, Lucio; Cerisoli, Francesco; Frati, Giacomo; Gaetani, Roberto; Chimenti, Isotta; Forte, Elvira; Cassinelli, Letizia; Spinardi, Laura; Altomare, Claudia; Kizana, Eddy; Giacomello, Alessandro; Messina, Elisa; Ottolenghi, Sergio; Magli, Maria Cristina

2011-01-01

320

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

PubMed Central

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

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

2014-01-01

321

Dendritic Cell-Mediated In Vivo Bone Resorption  

PubMed Central

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo, de novo osteoclastogenesis is observed but it is currently unknown whether, besides increased osteoclast differentiation from undifferentiated precursors, other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study, an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment, the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally, chromosomal painting identified nuclei from female DCs, previously injected into a male recipient, among the nuclei of giant cells at sites of osteolysis. Finally, osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r–/– mice, which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether, our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. PMID:20581147

Maitra, Radhashree; Follenzi, Antonia; Yaghoobian, Arash; Montagna, Cristina; Merlin, Simone; Cannizzo, Elvira S.; Hardin, John A.; Cobelli, Neil; Stanley, E. Richard; Santambrogio, Laura

2013-01-01

322

Repair of orbital bone defects in canines using grafts of enriched autologous bone marrow stromal cells  

PubMed Central

Backgroud Bone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to explore the feasibility of tissue-engineered bone constructed using bone marrow stromal cells (BMSCs) that were rapidly isolated and concentrated from bone marrow (BM) by the red cell lysis method, then combined with ?-tricalcium phosphate (?-TCP) to create grafts used to restore orbital bone defects in canines. Methods In the experimental group, grafts were constructed using BMSCs obtained by red cell lysis from 20 ml bone marrow, combined with ?-TCP and BM via the custom-made stem cell-scaffold device, then used to repair 10 mm diameter medial orbital wall bony defects in canines. Results were compared with those in groups grafted with BM/?-TCP or ?-TCP alone, or with defects left untreated as controls. The enrichment of BMSCs and nucleated cells (NCs) in the graft was calculated from the number in untreated bone marrow and in suspensions after red cell lysis. Spiral computed tomography (CT) scans were performed 1, 4, 12 and 24 weeks after implantation in all groups. Gross examination, micro-CT and histological measurements were performed 24 weeks after surgery. The results were analyzed to evaluate the efficacy of bone repair. Results The number of NCs and of colony-forming units within the scaffolds were increased 54.8 times and 53.4 times, respectively, compared with untreated bone marrow. In the BMSC-BM/?-TCP group, CT examination revealed that the scaffolds were gradually absorbed and the bony defects were restored. Micro-CT and histological examination confirmed that the implantations led to good repair of the defects, with 6 out 8 orbital defects completely restored in the experimental group, while by contrast, the grafts in the control groups did not fully repair the bony defects, a difference which was statistically significant (p?bone, constructed using BMSCs isolated by red cell lysis of BM, can restore critical-sized orbital wall defects in canines. PMID:24886296

2014-01-01

323

Interaction of progenitor bone cells with different surface modifications of titanium implant.  

PubMed

Changes in the physical and chemical properties of Ti surfaces can be attributed to cell performance, which improves surface biocompatibility. The cell proliferation, mineralization ability, and gene expression of progenitor bone cells (D1 cell) were compared on five different Ti surfaces, namely, mechanical grinding (M), electrochemical modification through potentiostatic anodization (ECH), sandblasting and acid etching (SLA), sandblasting, hydrogen peroxide treatment, and heating (SAOH), and sandblasting, alkali heating, and etching (SMART). SAOH treatment produced the most hydrophilic surface, whereas SLA produced the most hydrophobic surface. Cell activity indicated that SLA and SMART produced significantly rougher surfaces and promoted D1 cell attachment within 1 day of culturing, whereas SAOH treatment produced moderate roughness (Ra=1.26?m) and accelerated the D1 cell proliferation up to 7 days after culturing. The ECH surface significantly promoted alkaline phosphatase (ALP) expression and osteocalcin (OCN) secretion in the D1 cells compared with the other surface groups. The ECH and SMART-treated Ti surfaces resulted in maximum ALP and OCN expressions during the D1 cell culture. SLA, SAOH, and SMART substrate surfaces were rougher and exhibited better cell metabolic responses during the early stage of cell attachment, proliferation, and morphologic expressions within 1 day of D1 cell culture. The D1 cells cultured on the ECH and SMART substrates exhibited higher differentiation, and higher ALP and OCN expressions after 10 days of culture. Thus, the ECH and SMART treatments promote better ability of cell mineralization in vitro, which demonstrate their great potential for clinical use. PMID:24582253

Chen, Wen-Cheng; Chen, Ya-Shun; Ko, Chia-Ling; Lin, Yi; Kuo, Tzu-Huang; Kuo, Hsien-Nan

2014-04-01

324

High expression of leptin by human bone marrow adipocytes in primary culture  

Microsoft Academic Search

Adipocytes participate in the microen- vironment of the bone marrow (BM), but their exact role remains to be determined. It has recently been shown that leptin, a hormone secreted from extra- medullary adipocytes, could be involved in hemato- poiesis. Therefore we have developed a primary culture system of human BM adipocytes to character- ize their differentiation and determine whether leptin

PATRICK LAHARRAGUE; DOMINIQUE LARROUY; ANNE-MARIE FONTANILLES; NATHALIE TRUEL; ARTHUR CAMPFIELD; RENATA TENENBAUM; JEAN GALITZKY; JOEL X. CORBERAND; LUC PENICAUD; LOUIS CASTEILLA

325

Effect of Cyclic Strain on Cardiomyogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells  

Microsoft Academic Search

Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into

Yan Huang; Lisha Zheng; Xianghui Gong; Xiaoling Jia; Wei Song; Meili Liu; Yubo Fan

2012-01-01

326

Accelerated bone mass senescence after hematopoietic stem cell transplantation.  

PubMed

Osteoporosis and avascular necrosis (AVN) are long-lasting and debilitating complications of hematopoietic stem cell transplantation (HSCT). We describe the magnitude of bone loss, AVN and impairment in osteogenic cell compartment following autologous (auto) and allogeneic (allo) HSCT, through the retrospective bone damage revaluation of 100 (50 auto- and 50 allo-HSCT) long-term survivors up to 15 years after transplant. Current treatment options for the management of these complications are also outlined. We found that auto- and allo-HSCT recipients show accelerated bone mineral loss and micro-architectural deterioration during the first years after transplant. Bone mass density (BMD) at the lumbar spine, but not at the femur neck, may improve in some patients after HSCT, suggesting more prolonged bone damage in cortical bone. Phalangeal BMD values remained low for even more years, suggesting persistent bone micro-architectural alterations after transplant. The incidence of AVN was higher in allo-HSCT recipients compared to auto-HSCT recipients. Steroid treatment length, but not its cumulative dose was associated with a higher incidence of bone loss. Allo-HSCT recipients affected by chronic graft versus host disease seem to be at greater risk of continuous bone loss and AVN development. Reduced BMD and higher incidence of AVN was partly related to a reduced regenerating capacity of the normal marrow osteogenic cell compartment. Our results suggest that all patients after auto-HSCT and allo-HSCT should be evaluated for their bone status and treated with anti-resorptive therapy as soon as abnormalities are detected. PMID:23905076

Serio, B; Pezzullo, L; Fontana, R; Annunziata, S; Rosamilio, R; Sessa, M; Giudice, V; Ferrara, I; Rocco, M; De Rosa, G; Ricci, P; Tauchmanovà, L; Montuori, N; Selleri, C

2013-01-01

327

Accelerated Bone Mass Senescence After Hematopoietic Stem Cell Transplantation  

PubMed Central

Osteoporosis and avascular necrosis (AVN) are long-lasting and debilitating complications of hematopoietic stem cell transplantation (HSCT). We describe the magnitude of bone loss, AVN and impairment in osteogenic cell compartment following autologous (auto) and allogeneic (allo) HSCT, through the retrospective bone damage revaluation of 100 (50 auto- and 50 allo-HSCT) long-term survivors up to 15 years after transplant. Current treatment options for the management of these complications are also outlined. We found that auto- and allo-HSCT recipients show accelerated bone mineral loss and micro-architectural deterioration during the first years after transplant. Bone mass density (BMD) at the lumbar spine, but not at the femur neck, may improve in some patients after HSCT, suggesting more prolonged bone damage in cortical bone. Phalangeal BMD values remained low for even more years, suggesting persistent bone micro-architectural alterations after transplant. The incidence of AVN was higher in allo-HSCT recipients compared to auto-HSCT recipients. Steroid treatment length, but not its cumulative dose was associated with a higher incidence of bone loss. Allo-HSCT recipients affected by chronic graft versus host disease seem to be at greater risk of continuous bone loss and AVN development. Reduced BMD and higher incidence of AVN was partly related to a reduced regenerating capacity of the normal marrow osteogenic cell compartment. Our results suggest that all patients after auto-HSCT and allo-HSCT should be evaluated for their bone status and treated with anti-resorptive therapy as soon as abnormalities are detected. PMID:23905076

Serio, B; Pezzullo, L; Fontana, R; Annunziata, S; Rosamilio, R; Sessa, M; Giudice, V; Ferrara, I; Rocco, M; De Rosa, G; Ricci, P; Tauchmanovà, L; Montuori, N; Selleri, C.

2013-01-01

328

Isolation and Culture of Epithelial Stem Cells  

PubMed Central

In the skin, epithelial stem cells in the hair follicle contribute not only to the generation of a new hair follicle with each hair cycle, but also to the repair of the epidermis during wound healing. When these stem cells are isolated and expanded in culture, they can give rise to hair follicles, sebaceous glands, and epidermis when combined with dermis and grafted back onto Nude mice. In this chapter, we provide a method for isolating hair follicle epithelial stem cells from the skin of adult mice using immunofluorescent labeling to allow for the specific purification of epithelial stem cells by fluorescence-activated cell sorting (FACS). Notably, this method relies exclusively on cell surface markers, making it suitable for use with any strain of mouse and at various stages of the hair cycle. We also provide a detailed protocol for culturing epithelial stem cells isolated by FACS, allowing for analysis using a wide variety of culture assays. Additionally, we provide notes on using cultured cells for specific applications, such as viral manipulation and grafting. These techniques should be useful for directly evaluating stem cell function in normal mice and in mice with skin defects. PMID:19089359

Nowak, Jonathan A.; Fuchs, Elaine

2009-01-01

329

Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells  

PubMed Central

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. PMID:25470000

Rath, Subha N.; Brandl, Andreas; Hiller, Daniel; Hoppe, Alexander; Gbureck, Uwe; Horch, Raymund E.; Boccaccini, Aldo R.; Kneser, Ulrich

2014-01-01

330

Isolation and culture of pulmonary endothelial cells.  

PubMed

Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan. PMID:6090112

Ryan, U S

1984-06-01

331

Adipose-Derived Mesenchymal Cells for Bone Regereneration: State of the Art  

PubMed Central

Adipose tissue represents a hot topic in regenerative medicine because of the tissue source abundance, the relatively easy retrieval, and the inherent biological properties of mesenchymal stem cells residing in its stroma. Adipose-derived mesenchymal stem cells (ASCs) are indeed multipotent somatic stem cells exhibiting growth kinetics and plasticity, proved to induce efficient tissue regeneration in several biomedical applications. A defined consensus for their isolation, classification, and characterization has been very recently achieved. In particular, bone tissue reconstruction and regeneration based on ASCs has emerged as a promising approach to restore structure and function of bone compromised by injury or disease. ASCs have been used in combination with osteoinductive biomaterial and/or osteogenic molecules, in either static or dynamic culture systems, to improve bone regeneration in several animal models. To date, few clinical trials on ASC-based bone reconstruction have been concluded and proved effective. The aim of this review is to dissect the state of the art on ASC use in bone regenerative applications in the attempt to provide a comprehensive coverage of the topics, from the basic laboratory to recent clinical applications. PMID:24307997

Cicione, Claudia; Bernardini, Camilla; Michetti, Fabrizio; Lattanzi, Wanda

2013-01-01

332

VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation.  

PubMed

Vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) have been widely used in the fields of tissue engineering and regenerative medicine to stimulate angiogenesis and bone formation. The goal of this study was to determine whether VEGF and BMP-2 are involved in the homing of bone marrow stem cells (BMSCs) for bone regeneration and to provide insights into their mechanism of action. The chemoattraction of BMSCs to VEGF and BMP-2 was analysed in vitro using a checkerboard assay. VEGF and BMP-2 stimulated the chemotaxis of BMSCs but not chemokinesis. In vivo, both VEGF and BMP-2 also have been confirmed to induce the homing of tail vein injected BMSCs to the site of silk scaffold subcutaneous implantation in nude mice. When the scaffolds were implanted in the rabbit skull defects, more SSEA+ mesenchymal stem cells were mobilised and homed to silk scaffolds containing VEGF and/or BMP-2. More importantly, autogenic BMSCs were reinjected via the ear vein after labelling with lenti-GFP, and the cells were detected to home to the defects and differentiate into endothelial cells and osteogenic cells induced by VEGF and BMP-2. Finally, perfusion with Microfil showed that initial angiogenesis was enhanced in tissue-engineered complexes containing VEGF. Observations based on µCT assay and histological study revealed that bone formation was accelerated on BMP-2-containing scaffolds. These findings support our hypothesis that the localised release of VEGF and BMP-2 promote bone regeneration, in part by facilitating the mobilisation of endogenous stem cells and directing the differentiation of these cells into endothelial and osteogenic lineages. PMID:24425156

Zhang, W; Zhu, C; Wu, Y; Ye, D; Wang, S; Zou, D; Zhang, X; Kaplan, D L; Jiang, X

2014-01-01

333

Characterization of Native and Recombinant Bone Sialoprotein: Delineation of the Mineral-Binding and Cell Adhesion Domains and Structural Analysis of the RGD Domain  

Microsoft Academic Search

Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize. Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd ' ' 2.6 3 10 29 M, perhaps the strongest affinity for this mineral of any of the matrix proteins.

JOHN T. STUBBS; KEITH P. MINTZ; EDWARD D. EANES; DENNIS A. TORCHIA; LARRY W. FISHER

1997-01-01

334

Bone marrow cells as precursors of the tumor stroma  

PubMed Central

Cancer is a systemic disease. Local and distant factors conspire to promote or inhibit tumorigenesis. The bone marrow is one important source of tumor promoting cells. These include the important mature and immature hematopoietic cells as well as circulating mesenchymal progenitors. Recruited bone marrow cells influence carcinogenesis at the primary site, within the lymphoreticular system and even presage metastasis through their recruitment to distant organs. In this review we focus on the origins and contribution of cancer-associated fibroblasts in tumorigenesis. Mesenchymal cells present an important opportunity for targeted cancer prevention and therapy. PMID:23499739

Worthley, Daniel L.; Si, Yiling; Quante, Michael; Churchill, Michael; Mukherjee, Siddhartha; Wang, Timothy C.

2014-01-01

335

ERR{alpha} regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells  

SciTech Connect

The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.

Rajalin, Ann-Marie; Pollock, Hanna [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland)] [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland); Aarnisalo, Piia, E-mail: piia.aarnisalo@helsinki.fi [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland) [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland); Department of Clinical Chemistry, University of Helsinki and Helsinki University Central Hospital (Finland)

2010-05-28

336

Mechanical unloading of bone in microgravity reduces mesenchymal and hematopoietic stem cell-mediated tissue regeneration.  

PubMed

Mechanical loading of mammalian tissues is a potent promoter of tissue growth and regeneration, whilst unloading in microgravity can cause reduced tissue regeneration, possibly through effects on stem cell tissue progenitors. To test the specific hypothesis that mechanical unloading alters differentiation of bone marrow mesenchymal and hematopoietic stem cell lineages, we studied cellular and molecular aspects of how bone marrow in the mouse proximal femur responds to unloading in microgravity. Trabecular and cortical endosteal bone surfaces in the femoral head underwent significant bone resorption in microgravity, enlarging the marrow cavity. Cells isolated from the femoral head marrow compartment showed significant down-regulation of gene expression markers for early mesenchymal and hematopoietic differentiation, including FUT1(-6.72), CSF2(-3.30), CD90(-3.33), PTPRC(-2.79), and GDF15(-2.45), but not stem cell markers, such as SOX2. At the cellular level, in situ histological analysis revealed decreased megakaryocyte numbers whilst erythrocytes were increased 2.33 fold. Furthermore, erythrocytes displayed elevated fucosylation and clustering adjacent to sinuses forming the marrow-blood barrier, possibly providing a mechanistic basis for explaining spaceflight anemia. Culture of isolated bone marrow cells immediately after microgravity exposure increased the marrow progenitor's potential for mesenchymal differentiation into in-vitro mineralized bone nodules, and hematopoietic differentiation into osteoclasts, suggesting an accumulation of undifferentiated progenitors during exposure to microgravity. These results support the idea that mechanical unloading of mammalian tissues in microgravity is a strong inhibitor of tissue growth and regeneration mechanisms, acting at the level of early mesenchymal and hematopoietic stem cell differentiation. PMID:25011075

Blaber, E A; Dvorochkin, N; Torres, M L; Yousuf, R; Burns, B P; Globus, R K; Almeida, E A C

2014-09-01

337

Human cell culture in a space bioreactor  

NASA Technical Reports Server (NTRS)

Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

Morrison, Dennis R.

1988-01-01

338

Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.  

PubMed

Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1001-1010, 2015. PMID:24889783

Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

2015-03-01

339

Transferring isolated mitochondria into tissue culture cells  

PubMed Central

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained. PMID:22753025

Yang, Yi-Wei; Koob, Michael D.

2012-01-01

340

Calculator programmed assistance in making cell cultures  

Microsoft Academic Search

Summary A calculator program and instructions are presented for facilitating the preparation of single cell suspensions to be used in cell culture systems. The program has been written for the Texas Instruments Inc. TI-59 programmable calculator with printer. The instructions provided are intended for the novice with no previous programming experience and only a basic understanding of the calculator operation.

J. C. Bullaro

1980-01-01

341

Effect and clinical implications of the low-energy diode laser on bone cell proliferation.  

PubMed

Laser is a simple, noninvasive technique that has proven useful for treating damaged tissue. However, its effects on bone regeneration and the mechanisms involved are poorly understood. The objective of this study was to evaluate the effects on MG-63 cell proliferation of application of a pulsed diode laser (Ezlase) of 940 nm at low energy levels. After 24 hr of culture, osteoblasts underwent pulsed laser radiation at 0.5, 1, 1.5, and 2 W and fluences of 1-5 J. A control group was not irradiated. After the treatment, cells were incubated for 24 hr, and cell proliferation was analyzed using a spectrophotometric measure of cell respiration (MTT assay). Results were expressed as percentage proliferation versus controls. At 24-hr culture, cell proliferation was increased in laser-treated cells at intensities of 0.5, 1, and 1.5 W/cm(2) versus controls; the energy density was positively correlated with cell growth, which reached a peak at 3 J and decreased at higher fluences. The use of pulsed low-level laser with low-energy density range thus appears to exert a biostimulatory effect on bone tissue. Although the data on cell proliferation are robust, in-depth investigation is required into the effect of these irradiation doses on other cell parameters. The present findings demonstrate that laser therapy could be highly useful in tissue regeneration in different clinical settings, including nursing, physical therapy, dentistry, and traumatology. PMID:23559459

Huertas, Rosa Medina; Luna-Bertos, Elvira De; Ramos-Torrecillas, Javier; Leyva, Francisco Medina; Ruiz, Concepción; García-Martínez, Olga

2014-04-01

342

Noninvasive Real-Time Monitoring by AlamarBlue® During In Vitro Culture of Three-Dimensional Tissue-Engineered Bone Constructs  

PubMed Central

Bone tissue engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and nonfunctional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop noninvasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the nontoxic alamarBlue® (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was evaluated not only in static cultures, but also in dynamic cultures in a perfusion bioreactor setup. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a noninvasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence, the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct. PMID:23327780

Zhou, Xiaohua; Holsbeeks, Inge; Impens, Saartje; Sonnaert, Maarten; Bloemen, Veerle; Luyten, Frank

2013-01-01

343

Stem cells and bone diseases: new tools, new perspective.  

PubMed

Postnatal skeletal stem cells are a unique class of progenitors with biological properties that extend well beyond the limits of stemness as commonly defined. Skeletal stem cells sustain skeletal tissue homeostasis, organize and maintain the complex architectural structure of the bone marrow microenvironment and provide a niche for hematopoietic progenitor cells. The identification of stem cells in the human post-natal skeleton has profoundly changed our approach to the physiology and pathology of this system. Skeletal diseases have been long interpreted essentially in terms of defective function of differentiated cells and/or abnormal turnover of the matrix that they produce. The notion of a skeletal stem cell has brought forth multiple, novel concepts in skeletal biology that provide potential alternative concepts. At the same time, the recognition of the complex functions played by skeletal progenitors, such as the structural and functional organization of the bone marrow, has provided an innovative, unifying perspective for understanding bone and bone marrow changes simultaneously occurring in many disorders. Finally, the possibility to isolate and highly enrich for skeletal progenitors, enables us to reproduce perfectly normal or pathological organ miniatures. These, in turn, provide suitable models to investigate and manipulate the pathogenetic mechanisms of many genetic and non-genetic skeletal diseases. This article is part of a Special Issue entitled Stem cells and Bone. PMID:25240458

Riminucci, Mara; Remoli, Cristina; Robey, Pamela G; Bianco, Paolo

2015-01-01

344

Isolating phagosomes from tissue culture cells.  

PubMed

Phagocytosis is the process by which receptors at the plasma membrane are used to engulf a particle such as a bacterium, parasite, or dead cell. Phagosomes can be isolated from tissue culture cells by various centrifugation methods, including the use of differential density gradients or sucrose step gradients, but these methods are time-consuming or otherwise difficult. We describe here a protocol that avoids centrifugation and relies instead on the uptake of magnetic beads to rapidly isolate the phagosomal compartment from tissue culture cells. PMID:25447278

Pryor, Paul R; Rofe, Adam P

2014-12-01

345

Circadian gene expression in cultured cells.  

PubMed

In mammals, circadian oscillators not only exist in specialized neurons of the suprachiasmatic nucleus, but in almost all peripheral cell types. These oscillators are operative even in established fibroblast cell lines, such as Rat-1 cells or NIH3T3 cells, and in primary fibroblasts from mouse embryos or adult animals. This can be demonstrated by treating such cells for a short time period with high concentrations of serum or chemicals that activate a large number of known signaling pathways. The possibility of studying circadian rhythms in cultured cells should facilitate the biochemical and genetic dissection of the circadian clockwork and should promote the discovery of new clock components. PMID:15817311

Nagoshi, Emi; Brown, Steven A; Dibner, Charna; Kornmann, Benoît; Schibler, Ueli

2005-01-01

346

Cell culture experiments planned for the space bioreactor  

NASA Technical Reports Server (NTRS)

Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

Morrison, Dennis R.; Cross, John H.

1987-01-01

347

Cell-free and cell-based approaches for bone regeneration  

Microsoft Academic Search

The clinical augmentation of bone currently involves the use of autogenous or allogeneic bone grafts and synthetic materials, all of which are associated with limitations. Research on the safe enhancement of bone formation concerns the potential value of scaffolds, stem cells, gene therapy, and chemical and mechanical signals. Optimal scaffolds are engineered to provide mechanical stability while supporting osteogenesis, osteoconduction

Ericka M. Bueno; Julie Glowacki

2009-01-01

348

Tumor-host cell interactions in the bone disease of myeloma  

PubMed Central

Multiple myeloma is a hematological malignancy that is associated with the development of a destructive osteolytic bone disease, which is a major cause of morbidity for patients with myeloma. Interactions between myeloma cells and cells of the bone marrow microenvironment promote both tumor growth and survival and bone destruction, and the osteolytic bone disease is now recognized as a contributing component to tumor progression. Since myeloma bone disease is associated with both an increase in osteoclastic bone resorption and a suppression of osteoblastic bone formation, research to date has largely focused upon the role of the osteoclast and osteoblast. However, it is now clear that other cell types within the bone marrow, including cells of the immune system, mesenchymal stem cells and bone marrow stromal cells, can contribute to the development of myeloma bone disease. This review discusses the cellular mechanisms and potential therapeutic targets that have been implicated in myeloma bone disease. PMID:20615487

Fowler, Jessica A.; Edwards, Claire M.; Croucher, Peter I.

2010-01-01

349

Characteristics of cardiac cell cultures derived from human myocardial explants.  

PubMed

Primary cell cultures derived from human myocardial explants were obtained and characterized. The explant cultures contained cardiac stem cells (c-kit(+); ? 4%), microvascular cells (endothelial cells and pericytes), fibroblasts, and myofibroblasts. It was demonstrated that culturing of cardiac cells in cardiospheres did not promote enrichment of the cell culture with stem cells. MACS-sorted c-kit(+) cells from the explant culture were characterized by limited proliferative capacity and were capable of cardiomyogenic differentiation. The presence of microvascular cells determined general angiogenic potential of the culture. PMID:24319709

Pavlova, S V; Perovskii, P P; Chepeleva, E V; Malakhova, A A; Dement'eva, E V; Pokushalov, E A; Sukhikh, G T; Zakiyan, S M

2013-11-01

350

Paracrine Interactions between Mesenchymal Stem Cells Affect Substrate Driven Differentiation toward Tendon and Bone Phenotypes  

PubMed Central

We investigated substrate dependent paracrine signaling between subpopulations of bone marrow stromal cells (BMSCs) that may affect the formation, or perhaps malformation, of the regenerating tendon to bone enthesis. Polyacrylamide substrates approximating the elastic modulus of tendon granulation tissue and the osteoid of healing bone (10–90 kPa) were functionalized with whole length fibronectin (Fn), type-I collagen (Col), or a mixed ligand solution (Fn/Col), and BMSCs were cultured in growth media alone or media supplemented with soluble Col or Fn. More rigid substrates with a narrow mechanical gradient (70–90 kPa) robustly induced osteogenic cell differentiation when functionalized with either Col or Fn. On broader mechanical gradient substrates (with a linear elastic modulus gradient from 10–90 kPa), cell differentiation was markedly osteogenic on subregions of Fn functionalized substrates above 20 kPa, but osteogenic activity was inhibited on all subregions of Col substrates. Osteogenic behavior was not observed when cells were cultured on Fn substrates if Col was present either in the media or on the substrate (Fn/Col). Tenogenic differentiation markers were observed only on Col substrates with moderate rigidity (?30–50 kPa). Tenogenic differentiation was unaltered by soluble or substrate bound Fn. Co-culture of narrow gradient subsections revealed that any inclusion of tenogenic substrates (30–50 kPa, Col), caused otherwise osteogenic substrates to not develop markers of osteogenic differentiation, while increasing cell proliferation. These apparently paracrine effects could be mediated by bone morphogenetic protein-2 (BMP-2), as first confirmed by gene-level expression of BMP-2 and the transcription factor Smad8, and verified by BMP-2 media supplementation at levels similar to observed cell-secreted concentrations, which arrested osteogenic differentiation in 14 day cultures. Thus, cell instructive biomaterials with engineered mechanical and biochemical properties represent potentially powerful tools for directing BMSC differentiation to tendon and bone, however paracrine signals from tenogenic cells may delay osteogenesis at the healing enthesis. PMID:22355373

Sharma, Ram I.; Snedeker, Jess G.

2012-01-01

351

Encapsulated dental-derived mesenchymal stem cells in an injectable and biodegradable scaffold for applications in bone tissue engineering.  

PubMed

Bone grafts are currently the major family of treatment options in modern reconstructive dentistry. As an alternative, stem cell-scaffold constructs seem to hold promise for bone tissue engineering. However, the feasibility of encapsulating dental-derived mesenchymal stem cells in scaffold biomaterials such as alginate hydrogel remains to be tested. The objectives of this study were, therefore, to: (1) develop an injectable scaffold based on oxidized alginate microbeads encapsulating periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs); and (2) investigate the cell viability and osteogenic differentiation of the stem cells in the microbeads both in vitro and in vivo. Microbeads with diameters of 1?±?0.1 mm were fabricated with 2 × 10(6) stem cells/mL of alginate. Microbeads containing PDLSCs, GMSCs, and human bone marrow mesenchymal stem cells as a positive control were implanted subcutaneously and ectopic bone formation was analyzed by micro CT and histological analysis at 8-weeks postimplantation. The encapsulated stem cells remained viable after 4 weeks of culturing in osteo-differentiating induction medium. Scanning electron microscopy and X-ray diffraction results confirmed that apatitic mineral was deposited by the stem cells. In vivo, ectopic mineralization was observed inside and around the implanted microbeads containing the immobilized stem cells. These findings demonstrate for the first time that immobilization of PDLSCs and GMSCs in alginate microbeads provides a promising strategy for bone tissue engineering. PMID:23983201

Moshaverinia, Alireza; Chen, Chider; Akiyama, Kentaro; Xu, Xingtian; Chee, Winston W L; Schricker, Scott R; Shi, Songtao

2013-11-01

352

Osteopontin is an argentophilic protein in the bone matrix and in cells of kidney convoluted tubules  

E-print Network

sialoprotein -BSP, osteocalcine, bone morphogenetic proteins ) , . Numerous other proteins are elaboratedOsteopontin is an argentophilic protein in the bone matrix and in cells of kidney convoluted distribution. Human bone and kidney sections, benign and lymphomatous pleural effusions were processed

Paris-Sud XI, Université de

353

Development of a complex bone tissue culture system based on cellulose nanowhisker mechanical strain.  

PubMed

In bone tissue engineering, scaffolds have been investigated for their ability to support osteoblast growth and differentiation for recovery of damaged bones. Tunicate cellulose nanowhisker (CNW) film and mechanical strain were assessed for their suitability for osteoblasts. In this study, sulfuric acid hydrolysis extraction of tunicates integuments was conducted to obtain CNWs, which were found to be acceptable for adhering, growing, and differentiating osteoblasts without cytotoxicity. Mechanical stress enhanced osteoblast differentiation, and cell survival rate was recovered at around day 3, although there was a slight increase in cell death at day 1 after stimulation. We also found that intracellular flux of calcium ion was related to increased differentiation of CNWs under mechanical stress. Overall, we demonstrated the suitability of tunicate CNWs as a scaffold for bone tissue engineering and developed a complex system based on CNW for osteoblast growth and differentiation that will be useful for bone substitute fabrication. PMID:25454753

Kim, Dae Seung; Jung, Sang-Myung; Yoon, Gwang Heum; Lee, Hoo Cheol; Shin, Hwa Sung

2014-11-01

354

Canine coronavirus induces apoptosis in cultured cells  

Microsoft Academic Search

Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups.In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown.We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in

A. Ruggieri; L. Di Trani; I. Gatto; M. Franco; E. Vignolo; B. Bedini; G. Elia; C. Buonavoglia

2007-01-01

355

N-acetyl cysteine prevents polymethyl methacrylate bone cement extract-induced cell death and functional suppression of rat primary osteoblasts.  

PubMed

This study examines the cytotoxicity of bone cement extract to osteoblasts and the potential detoxification and restoration of osteoblastic function by an antioxidant amino acid, N-acetyl cysteine (NAC). The osteoblastic cells derived from rat femurs were cultured with extract from polymethyl methacrylate (PMMA)-based bone cement. The calcein and ethidium homodimer staining of the cells after 24-h incubation showed that 23.0% of the cells were dead in the culture with bone cement extract, while the addition of 5 mM NAC into the culture reduced the percentage to 4.3%. Annexin V and propidium iodide-based flow cytometric analysis also revealed that the apoptotic cells present at 15.8% in the culture with bone cement extract was reduced to 2.4% in the culture cotreated with bone cement extract and NAC. Severely suppressed alkaline phosphatase activity and matrix mineralization in the culture with bone cement extract (reduced to 10% and 5%, respectively, compared with the control culture) were restored to a normal level when treated with 5 mM NAC. The bone cement extract-induced, downregulated expression of osteoblastic genes, such as alkaline phosphatase, collagen I, and osteocalcin, was also restored to the baseline level by cotreatment with NAC. The data indicated that the addition of NAC into acrylic bone cement extract remarkably ameliorated the cytotoxicity to osteoblasts and restored their phenotype and function to a biologically significant degree, suggesting the potential usefulness of NAC in developing more biocompatible acrylic bone cement. PMID:19189384

Aita, Hideki; Tsukimura, Naoki; Yamada, Masahiro; Hori, Norio; Kubo, Katsutoshi; Sato, Naoko; Maeda, Hatsuhiko; Kimoto, Katsuhiko; Ogawa, Takahiro

2010-01-01

356

Effect of Intra-Medullar and Intra-Venous Infusions of Mesenchymal Stem Cells on Cell Engraftment by In-Vivo Cell Tracking and Osteoinductivity in Rabbit Long Bones: A Pilot Study  

PubMed Central

Objective Stem cell therapy can be an efficacious treatment option for bone fragility disorders (eg, osteogenesis imperfecta, disuse osteopenia, and osteoporosis), and successful cell therapy application may be dependent on optimal cell engraftment in target bones. The objective of this study was to compare the efficiency of intra-medullar and intra-venous delivery of mesenchymal stem cells (MSC) to improve cell engraftment rate, bone mineral density, and micro-architecture. Methods By using six healthy juvenile New Zealand White rabbits, MSC were isolated from cancellous bone harvests and confirmed to have osteogenic capacity by inducing ectopic bone formation. The MSC were cultured, transduced by foamy viral vectors with marker genes for in vivo cell tracking, and expanded. All rabbits had one randomly selected limb receive intra-medullar infusion of 3×107 to 1×108 autologous MSC in the distal femur or the distal femur and proximal tibia. Two of six rabbits also received an intra-venous MSC infusion. At 28 days, MSC bone engraftment was assessed by PCR and the bone density and microstructure assessed by computed tomography and histomorphometry. Results The intra-medullar-infused MSC were detected in epiphysis or diaphysis of the distal femurs and/or proximal tibiae. Infused MSC comprised 0.01 to 0.3% of all cells in the bone tissues. The intra-venous-infused MSC were not detected in any location. Neither intra-medullar nor intra-venous MSC infusion altered bone volume, bone mineral density, or cortical bone porosity/thickness. Systemic biodistribution of intra-medullar-infused MSC was not evident. Conclusions Our results indicated that intra-medullar infusion can be an effective cell delivery route for stem cell therapy potentially for orthopedic disorders, in preference to systemic administration. Further research is warranted to demonstrate an efficacy of intra-medullar MSC infusion on bone density and micro-architecture using animal models of bone disorders. PMID:25520900

Ishihara, Akikazu; Ohmine, Ken; Weisbrode, Steve E; Bertone, Alicia L

2014-01-01

357

Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells  

SciTech Connect

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

Montesano, Roberto [Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Rue-Michel-Servet, 1, CH-1211 Geneva 4 (Switzerland)], E-mail: Roberto.Montesano@unige.ch; Sarkoezi, Rita; Schramek, Herbert [Department of Internal Medicine IV, Nephrology and Hypertensiology, Innsbruck Medical University, Innsbruck (Austria)

2008-09-12

358

Potentiation of osteoclastogenesis by adipogenic conversion of bone marrow-derived mesenchymal stem cells.  

PubMed

Bone marrow-derived mesenchymal stem cells (BMSCs) are the indispensable component of the bone marrow, being the common precursors for adipocytes and osteoblasts. We show here that adipogenic differentiation resulted in increase in the production of adipocyte markers, such as adiponectin,fatty-acid binding proteins (FABP4), peroxisome proliferator-activated receptor ? (PPAR?), as well as the receptor activator of nuclear-?B ligand (RANKL). Co-culture of osteoclast precursors (OCPs) with BMSCs-derived adipocytes significantly enhanced osteoclast differentiation with low-dose RANKL, whose levels alone could not promote osteoclastogenesis. These results demonstrate for the first time that adipogenic differentiation of BMSCs plays a pivotal role in maintaining bone homeostasis. PMID:24759183

Mori, Keisuke; Suzuki, Keiji; Hozumi, Akira; Goto, Hisataka; Tomita, Masato; Koseki, Hironobu; Yamashita, Shunichi; Osaki, Makoto

2014-01-01

359

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress  

SciTech Connect

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

Tang, C.-H., E-mail: chtang@mail.cmu.edu.t [Department of Pharmacology, China Medical University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan (China); Chiu, Y.-C. [Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan (China); Department of Orthopaedics, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Nursing, Hungkuang University, Taichung County, Taiwan (China); Huang, C.-F. [School of Chinese Medicine, China Medical University, Taichung, Taiwan (China); Chen, Y.-W. [Department of Physiology, China Medical University, Taichung, Taiwan (China); Chen, P.-C. [Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China)

2009-12-01

360

Adult bone marrow stem cells for cell and gene therapies: implications for greater use.  

PubMed

There is excitement generated almost daily about the possible uses of stem cells to treat human disease. Much of the interest of late is generated by embryonic stem cells (ESCs). As exciting as ESCs may be, they are quite controversial for moral reasons, given their source. They are also scientifically controversial since they are much less well understood than the original, long-standing, and clinically successful hematopoietic stem cell (HSC). HSCs have the distinct advantage of being reasonably well characterized and have been proven in the clinic. They can be isolated by simple procedures directly from the bone marrow or from peripheral blood after being stimulated (mobilized). They can then be manipulated and delivered to a patient, often producing a cure. Their biology provides the paradigm by which all other stem cells are judged, and they have little in the way of moral controversy surrounding them given they are isolated from adults who have consented to the procedure. Another putative stem cell has gained momentum in the last few years; the mesenchymal stem cell (MSC). MSCs appear to have much in common with HSCs. They were originally characterized from bone marrow, are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. Unlike HSCs, they have not yet been definitively shown to function as stem cells, despite their ability to differentiate into various mesenchymal cell types under the right culture conditions. Still, there is mounting evidence these cells may be useful, if not as true stem cells then at least as vehicles for emerging cell and gene therapies, especially in the field of tissue engineering. While this is an important endpoint, it is more important to thoroughly understand stem cell biology. That understanding can then be applied toward the ultimate goal of using these cells not just for various forms of therapy, but rather as a tool to discover the mechanisms and means to bring about directed repair and regeneration of damaged or diseased tissues and organs. The excitement of HSCs and MSCs has been muted somewhat by the excitement surrounding ESCs, primarily due to the fact HSCs and MSCs are viewed as limited to specific cell types while ESCs could potentially be applied to any cell type. Recent information indicates HSCs, MSCs, and other cells in general may have more universal differentiation abilities than previously thought. PMID:12046846

Ballas, Christopher B; Zielske, Steven P; Gerson, Stanton L

2002-01-01

361

Platelet-Derived Growth Factor and Spatiotemporal Cues Induce Development of Vascularized Bone Tissue by Adipose-Derived Stem Cells  

PubMed Central

Vasculature is essential to the functional integration of a tissue-engineered bone graft to enable sufficient nutrient delivery and viability after implantation. Native bone and vasculature develop through intimately coupled, tightly regulated spatiotemporal cell–cell signaling. The complexity of these developmental processes has been a challenge for tissue engineers to recapitulate, resulting in poor codevelopment of both bone and vasculature within a unified graft. To address this, we cultured adipose-derived stromal/stem cells (ASCs), a clinically relevant, single cell source that has been previously investigated for its ability to give rise to vascularized bone grafts, and studied the effects of initial spatial organization of cells, the temporal addition of growth factors, and the presence of exogenous platelet-derived growth factor-BB (PDGF-BB) on the codevelopment of bone and vascular tissue structures. Human ASCs were aggregated into multicellular spheroids via the hanging drop method before encapsulation and subsequent outgrowth in fibrin gels. Cellular aggregation substantially increased vascular network density, interconnectivity, and pericyte coverage compared to monodispersed cultures. To form robust vessel networks, it was essential to culture ASCs in a purely vasculogenic medium for at least 8 days before the addition of osteogenic cues. Physiologically relevant concentrations of exogenous PDGF-BB (20?ng/mL) substantially enhanced both vascular network stability and osteogenic differentiation. Comparisons with the bone morphogenetic protein-2, another pro-osteogenic and proangiogenic growth factor, indicated that this potential to couple the formation of both lineages might be unique to PDGF-BB. Furthermore, the resulting tissue structure demonstrated the close association of mineral deposits with pre-existing vascular structures that have been described for developing tissues. This combination of a single cell source with a potent induction factor used at physiological concentrations can provide a clinically relevant approach to engineering highly vascularized bone grafts. PMID:23582144

Hutton, Daphne L.; Moore, Erika M.; Gimble, Jeffrey M.

2013-01-01

362

Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration  

Microsoft Academic Search

This study examines the clinical relevance of tissue engineering integrating gene therapy and polymer science to bone regeneration. Bilateral maxillary defects (3 × 1.2 cm2) in 20 miniature swine were bridged with a bioresorbable internal splint. Constructs were created using ex vivo adenovirus bone morphogenetic protein (BMP)-2-mediated gene transfer to the expanded bone marrow mesenchymal stem cells (MSCs) 7 days

S C-N Chang; H L Chuang; Y R Chen; J K Chen; H-Y Chung; Y-L Lu; H-Y Lin; C-L Tai; J Lou; SC-N Chang

2003-01-01

363

Lysophosphatidic acid-induced chemotaxis of bone cells  

Microsoft Academic Search

Lysophosphatidic acid (LPA) is a platelet-derived bioactive lipid that is postulated to regulate wound healing. LPA activates G protein-coupled receptors to induce Ca2+ signaling in MC3T3-E1 pre-osteoblasts, and is a potent chemotactic stimulus for these cells. Since bone fracture healing requires the migration of osteoblast progenitors, we postulate that LPA is among the factors that stimulate bone repair. UMR 106-01

Sue A. Karagiosis; Lisa M. Masiello; Nikki Bollinger; Norm J. Karin

2006-01-01

364

Selective bone cell adhesion on formulations containing carbon nanofibers  

Microsoft Academic Search

Bone cell adhesion on novel carbon nanofibers and polycarbonate urethane\\/carbon nanofiber (PCU\\/CNF) composites is investigated in the present in vitro study. Carbon nanofibers have exceptional theoretical mechanical properties (such as high strength to weight ratios) that, along with possessing nanoscale fiber dimensions similar to crystalline hydroxyapatite found in physiological bone, suggest strong possibilities for use as an orthopedic\\/dental implant material.

Rachel L Price; Michael C Waid; Karen M Haberstroh; Thomas J Webster

2003-01-01

365

Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells.  

PubMed

Human amnion mesenchymal stem cells (HAMSCs) can be obtained from human amniotic membrane, a highly abundant and readily available tissue. HAMSC sources present fewer ethical issues, have low immunogenicity, anti-inflammatory properties, considerable advantageous characteristics, and are considered an attractive potential treatment material in the field of regenerative medicine. We used a co-culture system to determine whether HAMSCs could promote osteogenesis in human bone marrow mesenchymal stem cells (HBMSCs). We isolated HAMSCs from discarded amnion samples and collected them using pancreatin/collagenase digestion. We cultured HAMSCs and HBMSCSs in basal medium. Activity of alkaline phosphatase (ALP), an early osteogenesis marker, was increased in the co-culture system compared to the control single cultures, which we also confirmed by ALP staining. We used immunofluorescence testing to investigate the effects of co-culturing with HAMSCs on HBMSC proliferation, which revealed that the co-culturing enhanced EdU expression in HBMSCs. Western blotting and quantitative real-time PCR indicated that co-culturing promoted osteogenesis in HBMSCs. Furthermore, Alizarin red S staining revealed that extracellular matrix calcium levels in mineralized nodule formation produced by the co-cultures were higher than that in the controls. Using the same co-culture system, we further observed the effects of HAMSCs on osteogenic differentiation in primary osteoblasts by Western blotting, which better addressed the mechanism for HAMSCs in bone regeneration. The results showed HAMSCs are osteogenic and not only play a role in promoting HBMSC proliferation and osteogenic differentiation but also in osteoblasts, laying the foundation for new regenerative medicine methods. PMID:25432786

Wang, Yuli; Yin, Ying; Jiang, Fei; Chen, Ning

2015-02-01

366

Mast Cell-Activated Bone Marrow Mesenchymal Stromal Cells Regulate Proliferation and Lineage Commitment of CD34+ Progenitor Cells  

PubMed Central

Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34+ progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates BM to release these effector cells in increased numbers. We hypothesize that mast cells (MCs) may play a predominant role in this process. Objective: To examine the effect of IgE-activated MCs on BM mesenchymal stromal cells which regulate proliferation and differentiation of CD34+ progenitors. Methods: Primary MCs were derived from CD34+ precursors and activated with IgE/anti-IgE. BM mesenchymal stromal cells were co-cultured with CD34+ progenitor cells and stimulated with IL-1/TNF or IgE/anti-IgE-activated MCs in Transwell system. Results: BM mesenchymal stromal cells produce low level of thymic stromal lymphopoietin (TSLP) under steady state conditions, which is markedly increased by stimulation with proinflammatory cytokines IL-1 and TNF or IgE-activated MCs. The latter also triggers bone marrow-derived mesenchymal stromal cells production of G-CSF, and GM-CSF while inhibiting SDF-1. MC-activated mesenchymal stromal cells stimulate CD34+ cells to proliferate and to regulate their expression of early allergy-associated genes. Conclusion and Clinical Relevance: This in vitro study indicates that IgE-activated MCs trigger BM mesenchymal stromal cells to release TSLP and hematopoietic growth factors and to regulate the proliferation and lineage commitment of CD34+ precursor cells. The data predict that the effective inhibition of MCs should impair mobilization and accumulation of allergic effector cells and thereby reduce the severity of allergic diseases. PMID:24381572

Allakhverdi, Zoulfia; Comeau, Michael R.; Armant, Myriam; Agrawal, Rachana; Woodfolk, Judith A.; Sehmi, Roma; Howie, Karen J.; Gauvreau, Gail M.; Delespesse, Guy

2013-01-01

367

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds.  

PubMed

Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (?-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration. PMID:22250840

Ye, Xinhai; Yin, Xiaofan; Yang, Dawei; Tan, Jian; Liu, Guangpeng

2012-07-01

368

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair  

PubMed Central

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications. PMID:20451676

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

2010-01-01

369

Fluorous solvent for cell culture  

Microsoft Academic Search

Incubation of mouse melanoma B16 cells in fluorous solvents with low boiling point such as perfluoromethylcyclohexane, 1,1,1,3,3,3-hexafluoro-2-propanol, ethylpentafluoropropionate resulted in cell death. However, cells lived up to 2 days in fluorous alcohols such as 2,2,3,3,4,4,5,5-octafluoro-1-pentanol and 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanol with relatively higher fluorine content. Remarkably, cells survived deprived of nutrition up to 4 days when incubated in 2,2,3,3,4,4,5,5,6,6,6-undecafluoro-1-hexanol or in 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptanol that

Maria Carmelita Z. Kasuya; Xiaonan Wen; Kenichi Hatanaka; Kageyasu Akashi

2011-01-01