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Microsoft Academic Search

A system is described for the formation of bone tissue in culture from isolated rat bone cells . The isolated bone cells were obtained from embryonic rat calvarium and perios- teum or from traumatized, lifted periosteum of young rats . The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional




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


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

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



Long-Term Culture of Human Bone Marrow Cells  

Microsoft Academic Search

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

Suzanne Gartner; Henry S. Kaplan



Microcarriers designed for cell culture and tissue engineering of bone.  


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

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



Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions  

Microsoft Academic Search

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs)\\u000a and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried\\u000a out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst

A. Crovace; A. Favia; L. Lacitignola; M. S. Di Comite; F. Staffieri; E. Francioso



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

SciTech Connect

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

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



In vitro generation of equine osteoclasts from bone marrow cells using a novel culture system  

Microsoft Academic Search

We report on preliminary results of a novel in vitro culture system designed to generate equine osteoclasts in large numbers. Osteoclast generation, as determined by the expression of tartrate resistant acid phosphatase (TRAP) and ability to resorb bone, was enhanced in equine bone marrow cultures supplemented with fibroblastic cell (L929) conditioned medium (L929-CM). Bone marrow was collected from a total

A. W. Gray; M. E. Davies; L. B. Jeffcott



The in vivo diffusion chamber technique for bone marrow or blood cell culture  

Microsoft Academic Search

Conclusions In vivo diffusion chamber culture technique provides a system that isolates implanted bone marrow or peripheral blood stem cells from host cells while allowing exchange of nutrients, waste produces, and presumed humoral stimulators and inhibitors of cell growth.

R. Willemze; R. I. Walker



Bone marrow monocytes in histiocytosis X acquire some phenotypic features of Langerhans cells in long term bone marrow cultures  

Microsoft Academic Search

Summary Bone marrow cells of a patient with Letterer-Siwe disease were cultured for three weeks in long-term bone marrow culture (LTBMC) conditions and examined at one-week intervals with a large panel of monoclonal antibodies by immunohistochemistry and by the immunogold transmission electron microscopy (immunoTEM) technique. Although at diagnosis the bone marrow showed a slight increase of monocytes with a normal

R. Luksch; D. Soligo; A. Cerri; L. Fina; E. Berti; G. Lambertenghi Deliliers



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

PubMed Central

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

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



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



Characterization of conditioned medium of cultured bone marrow stromal cells  

Microsoft Academic Search

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

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



Characterization of osteoblast-like behavior of cultured bone marrow stromal cells on various polymer surfaces  

Microsoft Academic Search

The creation of novel bone substitutes requires a detailed understanding of the interaction between cells and materials. This study was designed to test certain polymers, specifically poly(caprolactone) (PCL), poly(D,L-lactic-co- glycolic acid) (PLGA), and combinations of these polymers for their ability to support bone marrow stromal cell prolif- eration and differentiation. Bone marrow stromal cells were cultured from New Zealand White

Jay W. Calvert; Kacey G. Marra; Lisa Cook; Prashant N. Kumta; Paul A. DiMilla; Lee E. Weiss



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

PubMed Central

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

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



Analysis of Cells Isolated from Bone Cultured on Collagen Gels and Polystyrene Culture Dishes.  

National Technical Information Service (NTIS)

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 feta...

K. Fletcher



Valproate reduces collagen and osteonectin in cultured bone cells.  


Valproate is a histone deacetylase (HDAC) inhibitor that was introduced more than 40 years ago and is commonly used to treat epilepsy and mood disorders. Its long-term side effects can include bone loss, although the exact mechanism for this is currently unknown. In a previous study, we used iTRAQ labelling and mass spectrometry to profile the effect of valproate on skin fibroblast cells. We found, for the first time, that valproate reduced the amount of two key bone proteins; collagen I and osteonectin (SPARC, BM-40) while over 1000 other proteins remained unchanged (Fuller et al., 2010). We now show that valproate treatment also reduces the protein levels of collagen I and osteonectin in the hFOB1.19 osteoblast-like cell line. Pro-collagen I was reduced by 48% and osteonectin by 25% after 24h exposure to a clinically-relevant concentration of valproate. Collagen I is the main protein component of bone matrix and osteonectin has a major role in bone development and mineralisation, so reduced levels may contribute to bone loss following long-term in vivo exposure to valproate. PMID:23906561

Humphrey, Emma L; Morris, Glenn E; Fuller, Heidi R



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

PubMed Central

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

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



Improved conditions to induce hepatocytes from rat bone marrow cells in culture.  


Recent studies have revealed that bone marrow cells can develop into hepatocytes by in vivo transplantation under certain circumstances. However, little is known about the mechanism of bone marrow cell differentiation into hepatocytes. It is important to determine suitable culture conditions in which bone marrow cells will be differentiated into hepatocytes not only for understanding differentiation mechanisms but also for efficient amplification of hepatocyte-progenitor cells of bone marrow origin, this being a prerequisite for potential therapeutic use. In the present study, we found that hepatocyte growth factor (HGF) receptor (c-Met)- and alpha-fetoprotein-expressing cells were present in adult rat bone marrow. We also found that these cells also express hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. Using an HGM medium with HGF and EGF, we succeeded in propagating hepatocyte-like cells induced from adult rat bone marrow in culture. These cells were immunocytochemically stained for albumin. By RT-PCR analysis of cultures containing the hepatocyte-like cells, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture therefore can be a useful resource for cell transplantation therapy for liver diseases. PMID:12379214

Miyazaki, Masahiro; Akiyama, Ichiro; Sakaguchi, Masakiyo; Nakashima, Emiko; Okada, Mayumi; Kataoka, Ken; Huh, Nam-ho



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

Microsoft Academic Search

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

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



Microscopy analysis of bone marrow-derived osteoprogenitor cells cultured on hydrogel 3-D scaffold.  


Bone marrow contains progenitor cells that are able to differentiate into several mesenchymal lineages, including bone. These cells may also provide a potential therapy for bone repair. The purpose of this study was to select the osteoprogenitor cell subpopulation from bone marrow-derived mesenchymal stem cells (MSCs) and to test the ability of a hydrogel scaffold to support growth and osteogenic differentiation. MSCs isolated from rat femur bone marrow were cultured in DMEM medium supplemented with antibiotics, FCS, and L-glutamine. Osteogenic supplements (dexamethasone, sodium beta-glycerophosphate, and ascorbic acid) were added for one, two or three weeks. A selective subpopulation of osteoprogenitor cells was identified by immunohistochemistry, general morphology, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Committed osteogenic cells were transferred to a 3-D hydrogel scaffold and cultured for an additional week. In standard culture, the osteoprogenitor cells formed cell clusters identified by Alizarin red S staining and by positive osteocalcin immunostaining. The number of osteoprogenitor cells, matrix synthesis, and mineralization increased gradually up to three weeks in culture. Mineral deposition in the matrix analyzed by EDS revealed the presence of calcium and phosphate ions at a Ca/P molar ratio of 1.73 in both the osteogenic cultures and the scaffold osteoprogenitor culture. Histological preparations revealed cell clusters within the hydrogel scaffold and SEM analysis revealed cell clusters attached to the scaffold surface. It is concluded that the hydrogel scaffold can support growth and differentiation of osteogenic cultures including mineralization and can potentially serve as a bone graft substitute containing committed osteoprogenitor cells. PMID:15880496

Srouji, S; Maurice, S; Livne, E



Repairing goat tibia segmental bone defect using scaffold cultured with mesenchymal stem cells.  


In this study, we investigated cellular biocompatibility in vitro and segmental bone defect repairing efficacy in vivo of a previously reported fibre-reinforced scaffold, nano-hydroxyapatite/collagen/poly (L-lactic acid) (PLLA)/chitin fibres (nHACP/CF). First, attachment, proliferation, and differentiation of the goat bone mesenchymal stem cells (GBMSCs) cultured on the nHACP/CF scaffolds were evaluated in vitro. The results showed that cells attached to the scaffolds well, and there was no significant difference in cell proliferation between cells on the scaffolds and cells on the polystyrene culture plates that were used as a control. The results also showed that alkaline phosphatase (ALP)/DNA of the cells cultured on the scaffolds was significantly higher than that on the control. The in vivo study compared the bone defect repairing efficacy of nHACP/CF scaffolds with that of autograft bone. Thirty-two adult male goats with 25-mm defects in their tibias at the same anatomic site were divided into four groups. The first group was implanted with the nHACP/CF with GBMSCs. The second group was implanted with autograft bone. The third group was implanted with the nHACP/CF. Nothing was implanted in the fourth group. Bone growth was evaluated by radiography, histology, and biomechanics. The results showed that although the nHACP/CF had new bone formation, it could not repair the defect fully while nHACP/CF with GBMSCs cultured and autograft bone could repair the segmental bone defect by 8 weeks after surgery, suggesting that nHACP/CF is an appropriate scaffold for bone tissue engineering. PMID:20336727

Liu, Xinhui; Li, Xiaoming; Fan, Yubo; Zhang, Guoping; Li, Dongmei; Dong, Wei; Sha, Ziyi; Yu, Xiaoguang; Feng, Qingling; Cui, Fuzhai; Watari, Fumio



Improved culture-based isolation of differentiating endothelial progenitor cells from mouse bone marrow mononuclear cells.  


Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse. PMID:22216102

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



Cell lines and primary cell cultures in the study of bone cell biology  

Microsoft Academic Search

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite and amorphous calcium phosphate crystals deposited in an organic matrix. Bone has two main functions. It forms a rigid skeleton and has a central role in calcium and phosphate homeostasis. The major cell types of bone are osteoblasts, osteoclasts and chondrocytes. In the laboratory,

Vicky Kartsogiannis; Kong Wah Ng



Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells.  

PubMed Central

Growth cartilage cells were isolated from the ribs of young rats and cultured at high cell density in Ham's F-12 medium supplemented with 10% fetal calf serum. During 7 days, glycosaminoglycans and proteoglycans were actively synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured and biosynthetically prelabeled with 35SO4(2-) in their glycosaminoglycans, bone marrow cells caused release of 35S-labeled material into the culture medium. Glycosaminoglycan was also released by addition of conditioned medium obtained from cultures of bone marrow cells or peritoneal macrophages to the growth cartilage cell cultures. Electron microscopic studies of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of apatite mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells or macrophages removed glycosaminoglycan or proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was initiated. From these findings, sequential culture of growth cartilage cells and bone marrow cells is promising as an experimental system for investigating the mechanism of the initial stage of endochondral ossification. Images

Suzuki, F; Takase, T; Takigawa, M; Uchida, A; Shimomura, Y



Bone marrow mononuclear cells protect neurons and modulate microglia in cell culture models of ischemic stroke.  


Although several studies have provided evidence for the therapeutic potential of bone marrow-derived mononuclear cells (MNCs) in animal models of stroke, the mechanisms underlying their benefits remain largely unknown. We have determined the neuroprotective potential of MNCs in primary neuronal cultures exposed to various injuries in vitro. Cortical neurons in culture were exposed to oxygen-glucose deprivation, hypoxia, or hydrogen peroxide, and cell death was assayed by MTT, caspase-3 activation or TUNEL labelling at 24 hrs. Cultures were randomized to cotreatment with MNC-derived supernatants or media before injury exposure. In separate experiments, macrophage or microglial cultures were exposed to lipopolypolysacharide (LPS) in the presence and absence of MNC-derived supernatants. Neuronal cultures were then exposed to conditioned media derived from activated macrophages or microglia. Cytokines from the supernantants of MNC cultures exposed to normoxia or hypoxia were also estimated by enzyme-linked immunosorbant assay (ELISA). MNC-derived supernatants attenuated neuronal death induced by OGD, hypoxia, hydrogen peroxide, and conditioned macrophage/microglial media and contain a number of trophic factors, including interleukin-10, insulin-like growth factor-1, vascular endothelial growth factor, and stromal cell-derived factor-1. MNCs provide broad neuroprotection against a variety of injuries relevant to stroke. PMID:20629187

Sharma, Sushil; Yang, Bing; Strong, Roger; Xi, XiaoPei; Brenneman, Miranda; Grotta, James C; Aronowski, Jaroslaw; Savitz, Sean I



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))



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

NASA Astrophysics Data System (ADS)

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

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



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

Microsoft Academic Search

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

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



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

PubMed Central

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

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



Estradiol synthesis and release in cultured female rat bone marrow stem cells.  


Bone marrow stem cells (BMSCs) have the capacity to differentiate into mature cell types of multiple tissues. Thus, they represent an alternative source for organ-specific cell replacement therapy in degenerative diseases. In this study, we demonstrated that female rat BMSCs could differentiate into steroidogenic cells with the capacity for de novo synthesis of Estradiol-17 ? (E2) under high glucose culture conditions with or without retinoic acid (RA). The cultured BMSCs could express the mRNA and protein for P450arom, the enzyme responsible for estrogen biosynthesis. Moreover, radioimmunoassay revealed that BMSCs cultured in the present culture system produced and secreted significant amounts of testosterone, androstenedione, and E2. In addition, RA promoted E2 secretion but did not affect the levels of androgen. These results indicate that BMSCs can synthesize and release E2 and may contribute to autologous transplantation therapy for estrogen deficiency. PMID:23484106

Zhang, Dalei; Yang, Bei; Zou, Weiying; Lu, Xiaying; Xiong, Mingdi; Wu, Lei; Wang, Jinglei; Gao, Junhong; Xu, Sifan; Zou, Ting



Estradiol Synthesis and Release in Cultured Female Rat Bone Marrow Stem Cells  

PubMed Central

Bone marrow stem cells (BMSCs) have the capacity to differentiate into mature cell types of multiple tissues. Thus, they represent an alternative source for organ-specific cell replacement therapy in degenerative diseases. In this study, we demonstrated that female rat BMSCs could differentiate into steroidogenic cells with the capacity for de novo synthesis of Estradiol-17? (E2) under high glucose culture conditions with or without retinoic acid (RA). The cultured BMSCs could express the mRNA and protein for P450arom, the enzyme responsible for estrogen biosynthesis. Moreover, radioimmunoassay revealed that BMSCs cultured in the present culture system produced and secreted significant amounts of testosterone, androstenedione, and E2. In addition, RA promoted E2 secretion but did not affect the levels of androgen. These results indicate that BMSCs can synthesize and release E2 and may contribute to autologous transplantation therapy for estrogen deficiency.

Zhang, Dalei; Yang, Bei; Zou, Weiying; Lu, Xiaying; Xiong, Mingdi; Wu, Lei; Wang, Jinglei; Gao, Junhong; Xu, Sifan; Zou, Ting



The genotoxic and cytotoxic activities of inorganic fluoride in cultured rat bone marrow cells  

Microsoft Academic Search

The effects of sodium and potassium fluoride (NaF and KF) at concentrations ranging from 10-7 to 10-2 M for 12, 24, or 36 h on cultured rat bone marrow cells have been studied with respect to cytotoxicity and induction of sister-chromatid exchanges (SCE). At the three exposure times, cell survival progressively decreased with increasing concentrations. Treatment with 10-2 M fluoride

A. M. Khalil; A. A. Da'dara



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.



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

PubMed Central

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

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



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


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


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



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

SciTech Connect

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

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



Desensitization of parathyroid hormone receptors on cultured bone cells  

SciTech Connect

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

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



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.



Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation  

Microsoft Academic Search

Tissue engineering of bone grafts with osteogenic progenitor cells such as adult mesenchymal stem cells (MSC) represents a\\u000a promising strategy for the treatment of large bone defects. The aim of this experimental study was to evaluate the osteogenic\\u000a potential of primary osteoblasts on MSCs in co-culture at different ratios. The co-cultures were treated with or without a\\u000a specific osteogenic induction

C. Csaki; U. Matis; A. Mobasheri; M. Shakibaei



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.

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



Three-dimensional culture of mouse bone marrow cells within a porous polymer scaffold: effects of oxygen concentration and stromal layer on expansion of haematopoietic progenitor cells.  


To establish an ex vivo expansion method of haematopoietic progenitor cells (HPCs) and erythroid cells, three-dimensional (3D) cultures of mouse bone marrow cells were performed, employing a porous polyvinyl formal (PVF) resin as a scaffold. In these cultures, the effects of oxygen concentration and co-cultures with stromal cells on the expansion of HPCs and erythroid cells were investigated. When bone marrow cells were cultured under 3D conditions, HPCs and erythroid cells expanded without supplementation of exogenous cytokines, irrespective of the presence of stromal cells. On the contrary, slight expansion of HPCs or erythroid cells was observed in monolayer cultures as controls, indicating that the 3D cultures using the PVF scaffold were far better in expanding HPCs and erythroid cells than the monolayer cultures. Under hypoxic conditions, bone marrow stromal cells allowed for a 3D culture of erythroid cells and HPCs at higher cell densities compared to cultures without stromal cells, and the duration of the expansion of HPCs and erythroid cells after initiating the 3D co-cultures was prolonged. The number of these cells increased throughout the culture period up to 3 weeks under hypoxic conditions, although the number decreased after 2 weeks under normoxic conditions. In conclusion, the 3D co-culture method of haematopoietic cells with stromal cells under hypoxic conditions was confirmed to be effective in expanding HPCs and erythroid cells, and this method seemed to be useful for developing an ex vivo expansion method for haematopoietic cells. PMID:20653040

Miyoshi, Hirotoshi; Murao, Mariko; Ohshima, Norio; Tun, Thein



Pre-culture period of mesenchymal stem cells in osteogenic media influences their in vivo bone forming potential  

Microsoft Academic Search

The objective of this study was to investigate if the in vitro pre-culture period in osteogenic media of rat mesenchymal stem cells (MSCs), influences their ability to regenerate bone when implanted in a critical size cranial defect. MSCs were harvested from the bone marrow of 6-8 weeks old male Fisher rats and expanded in vitro in osteogenic media for different

H. Castano-Izquierdo; J. Alvarez-Barreto; J. van den Dolder; J. A. Jansen; A. G. Mikos; V. I. Sikavitsas



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


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

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



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


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

Li, Xueyuan; Zhang, Yang; Qi, Guoxian



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

Microsoft Academic Search

Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem\\u000a cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis.\\u000a Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated\\u000a mineralized miromasses. Embryonic

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



Stromal cells in long-term murine bone marrow culture: FACS studies and origin of stromal cells in radiation chimeras  

SciTech Connect

Adherent layers from hematopoietically active long-term bone marrow cultures (LTBMC), incubated with fluorescent beads, were analyzed for autofluorescence and phagocytic ability, using a fluorescence-activated cell sorter (FACS). Four groups of cells were separated from the adherent layers, including a group of large polygonal fibroblastoid stromal cells. Long-term chimeras were made by lethal irradiation of CBA/Ca (CBA) and C57Bl6/J (B6) mice and repopulation with phosphoglycerate kinase (PGK-1) alloenzyme-congenic bone marrow cells. Hematopoietically active LTBMC were established from such chimeras, and donor and host contributions of FACS-sorted adherent-layer cells were measured. While macrophages and other hematopoietic cells were of donor origin, the fibroblastoid stromal cells were mainly or entirely host derived.

Lennon, J.E.; Micklem, H.S.



Evaluation of adult equine bone marrow- and adipose-derived progenitor cell chondrogenesis in hydrogel cultures.  


Bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived progenitor cells (ADPCs) are potential alternatives to autologous chondrocytes for cartilage resurfacing strategies. In this study, the chondrogenic potentials of these cell types were compared by quantifying neo-tissue synthesis and assaying gene expression and accumulation of extracellular matrix (ECM) components of cartilage. Adult equine progenitor cells encapsulated in agarose or self-assembling peptide hydrogels were cultured in the presence or absence of TGFbeta1 for 3 weeks. In BM-MSCs-seeded hydrogels, TGFbeta1 stimulated ECM synthesis and accumulation 3-41-fold relative to TGFbeta1-free culture. In ADPC cultures, TGFbeta1 stimulated a significant increase in ECM synthesis and accumulation in peptide (18-29-fold) but not agarose hydrogels. Chromatographic analysis of BM-MSC-seeded agarose and peptide hydrogels cultured in TGFbeta1 medium showed extensive synthesis of aggrecan-like proteoglycan monomers. ADPCs seeded in peptide hydrogel also synthesized aggrecan-like proteoglycans, although to a lesser extent than seen in BM-MSC hydrogels, whereas aggrecan-like proteoglycan synthesis in ADPC-seeded agarose was minimal. RT-PCR analysis of TGFbeta1 cultures showed detectable levels of type II collagen gene expression in BM-MSC but not ADPC cultures. Histological analysis of TGFbeta1-cultured peptide hydrogels showed the deposition of a continuous proteoglycan- and type II collagen rich ECM for BM-MSCs but not ADPCs. Therefore, this study showed both protein and gene expression evidence of superior chondrogenesis of BM-MSCs relative to ADPCs. PMID:17960654

Kisiday, John D; Kopesky, Paul W; Evans, Christopher H; Grodzinsky, Alan J; McIlwraith, C Wayne; Frisbie, David D



Bone formation by osteoblast-like cells in a three-dimensional cell culture  

Microsoft Academic Search

Summary  Cells of the clonal osteogenic cell line MC3T3-E1 were seeded onto a three-dimensional matrix of denatured collagen type 1\\u000a and cultured for a period of up to 8 weeks. Specimens were analyzed by histological, enzyme histochemical, immunocytochemical,\\u000a and ultrastructural methods and byin situ hybridization between day 7 and day 56 after seeding. In 56-day cultures, the MC3T3-E1 cells were arranged

M. Casser-Bette; A. B. Murray; E. I. Closs; V. Erfle; J. Schmidt



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

Microsoft Academic Search

This study was performed to establish a bone marrow stromal cell (BMSC)\\/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC\\/neuron co-culture

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



Novel starch-based scaffolds for bone tissue engineering: cytotoxicity, cell culture, and protein expression.  


Starch-based biomaterials and scaffolds have been proposed for several biomedical applications. In the present work new scaffolds based on a 50/50 (wt%) blend of corn starch/ethylene-vinyl alcohol (SEVA-C) were studied. These scaffolds were processed by a melt-based technology, which has been used before with other starch-based materials but never with SEVA-C. Scanning electron microscopy (SEM) observation showed that the developed porous structures were 60% porous with pore size between 200 and 900 microm and a reasonable degree of interconnectivity. Moreover, scaffolds presented a compressive modulus of 117.50 +/- 3.7 MPa and a compressive strength of 20.8 +/- 2.4 MPa. Cytotoxicity evaluation was performed according to ISO/EN 10993 part 5 guidelines, and revealed that the developed scaffolds were nontoxic and did not inhibit cell growth. Direct contact assays were also carried out by use of a cell line of human osteoblast-like cells (SaOS-2). Cells were seeded (3 x 10(5) per scaffold) and allowed to grow for 4 weeks at 37 degrees C, in a humidified atmosphere containing 5% CO(2). Total protein assay showed that the cells were able to grow for the 4 weeks of the experiment. These data were further confirmed by SEM. Moreover, a cell viability assay (MTS test) demonstrated that cells were perfectly viable after the 4 weeks of culture, showing the adequacy of the developed structure in supporting them. Finally, Western blot analysis revealed that osteopontin was being actively expressed by the cells, which, in association with collagen deposition observed by SEM, seems to indicate that bone extracellular matrix was being deposited. Consequently it is believed that starch-based scaffolds should be considered as an alternative for bone tissue-engineering applications in the near future. PMID:15165463

Salgado, A J; Coutinho, O P; Reis, R L


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



Cultured autologous bone marrow stem cells inhibit bony fusion in a rabbit model of posterolateral lumbar fusion with autologous bone graft.  


Mesenchymal stem cells (MSCs) have been isolated from various tissues and expanded in culture. MSCs add osteogenic potential to ceramic scaffolds when used together. A spinal fusion rabbit model was used to evaluate whether a pellet of cultured, autologous bone marrow MSCs (BMSCs) with osteogenic differentiation could increase the fusion rate when co-grafted with an autologous bone graft compared to autograft alone. Thirty rabbits were randomly assigned to two groups. Group 1 received bone autograft alone and Group 2 received bone autograft plus a pellet of cultured and differentiated BMSCs. Group 2 rabbits had a bone marrow puncture, after which the BMSC were cultured and osteoblastic differentiation was induced. BMSC cultures were obtained from 12 of 15 rabbits. The 27 rabbits underwent a bilateral, L4-L5 intertransverse fusion with an autograft and in Group 2 rabbits a pellet of differentiated BMSCs was added to the autograft. In Group 1, the fusion rate was 53% (8 of 15 rabbits) and in Group 2 the fusion rate was 0% (p<0.05). Adding differentiated BMSCs in a pellet without a scaffold not only failed to increase fusion rate, but completely inhibited bony growth. PMID:20171892

Urrutia, Julio; Mery, Pablo; Martínez, Rafael; Pizarro, Felipe; Apablaza, Daniel; Mardones, Rodrigo



Hydrogel/calcium phosphate composites require specific properties for three-dimensional culture of human bone mesenchymal cells.  


To provide multipotent cells with a three-dimensional environment closer to bone matrix, an engineered construct mimicking bone components has been designed and evaluated. A biocompatible hydrogel (silated hydroxypropylmethyl cellulose) was used as an extra-cellular matrix while biphasic calcium phosphate ceramic particles were used to replace mineralized matrix. Finally, human bone mesenchymal cells were cultured in three dimensions in the resulting constructs to study their cell viability, proliferation, interactions within the composites, and maintenance of their osteogenic potential. This approach resulted in homogeneous structures in which cells were viable and retained their osteoblastic differentiation potential. However, the cells did not proliferate nor colonize the constructs, possibly because of a lack of suitable interactions with their micro-environment. PMID:20152947

Sohier, J; Corre, P; Weiss, P; Layrolle, P



The impact of cell source, culture methodology, culture location, and individual donors on gene expression profiles of bone marrow-derived and adipose-derived stromal cells.  


Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells. PMID:23145933

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



Human bone cells in vitro.  


Human bone cell cultures were established by maintaining collagenase-treated bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 micrograms/ml) and 10 mM beta-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolism in vitro. PMID:2998572

Robey, P G; Termine, J D



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

PubMed Central

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

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



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.



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

Microsoft Academic Search

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

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



Morphogenesis and proliferation in mono- and organotypic co-cultures of primary human periodontal ligament fibroblasts and alveolar bone cells  

Microsoft Academic Search

Cells of the periodontal ligament and the alveolar bone lie in close vicinity in the periodontium. The goal of this study was to create an in vitro model to facilitate the study of the morphogenesis and proliferation of these two cell types under more in-vivo-like conditions. This was accomplished by the generation of organotypic co-cultures of primary human periodontal ligament

T. Reuther; A. Kohl; G. Komposch; P. Tomakidi



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


This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture. PMID:20378357

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



Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells  

Microsoft Academic Search

Bone-marrow-derived mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and differentiation into multiple\\u000a cell types. Accumulating preclinical and clinical evidence indicates that MSCs are good candidates to use as cell therapy\\u000a in many degenerative diseases. For MSC clinical applications, an adequate number of cells are necessary so an extensive expansion\\u000a is required. However, spontaneous immortalization and malignant transformation

Dana Foudah; Serena Redaelli; Elisabetta Donzelli; Angela Bentivegna; Mariarosaria Miloso; Leda Dalprà; Giovanni Tredici



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


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



Effect of electromagnetic fields on proliferation and differentiation of cultured mouse bone marrow mesenchymal stem cells  

Microsoft Academic Search

Summary  In order to study the effects of electromagnetic fields (EMFs) on proliferation, differentiation and intercellular cyclic\\u000a AMP (cAMP) in mouse bone marrow mesenchymal stem cells (MSCs)in vitro, the mouse bone MSCs were isolated and culturedin vitro. The third passage MSCs were divided into 4 groups and stimulated with EMFs. The cellular proliferation (MTT), the cellular\\u000a differentiation (alkaline phosphatase activity, ALP),

Wu Hua; Ren Kai; Zhao Wenchun; Ge Baojian; Peng Songlin



Differentiation of the rat myelomonocytic leukemia cell line c-WRT-7 by in vitro culture with the rat bone marrow preadipocyte cell line REC A16  

Microsoft Academic Search

Differentiation of the rat myelomonocytic leukemia cell line (c-WRT-7) was investigated, by co-culture with a rat embryonic bone marrow preadipose cell line (REC A16). Co-cultivation with REC A16, or with conditioned medium from REC A16 cultures (REC-CM), induced differentiation of c-WRT-7 cells to macrophages. A soluble factor(s) produced by REC A16 appeared to be responsible for the differentiation of c-WRT-7.

Takeshi Tanaka; Seiichi Okamura; Shigeru Yasumoto; Noritoshi Takeichi; Hiroshi Kobayashi; Yoshiyuki Niho



The cellular metabolism of lead and calcium: A kinetic analysis in cultured osteoclastic bone cells  

Microsoft Academic Search

Characterization of lead metabolism in bone is necessary to understand the role of skeletal lead, an endogenous source of this toxic metal, in the expression of adverse effects of lead intoxication in concert with external sources. Moreover, it has been postulated that an early manifestation of lead toxicity common to diverse cell types may be pertubations in intracellular calcium homeostasis.

J. F. Rosen; J. G. Pounds



Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture  

Microsoft Academic Search

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17?-estradiol (E2) on osteoblast-like cells was investigated by using mouse bone marrow cultures. Bone marrow cells were harvested from the shafts of femurs of 10-week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under

Q. Qu; M. Perälä-Heape; A. Kapanen; J. Dahllund; J. Salo; H. K. Väänänen; P. Härkönen



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

PubMed Central

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

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



Coculture Strategies in Bone Tissue Engineering: The Impact of Culture Conditions on Pluripotent Stem Cell Populations  

PubMed Central

The use of pluripotent stem cell populations for bone tissue regeneration provides many opportunities and challenges within the bone tissue engineering field. For example, coculture strategies have been utilized to mimic embryological development of bone tissue, and particularly the critical intercellular signaling pathways. While research in bone biology over the last 20 years has expanded our understanding of these intercellular signaling pathways, we still do not fully understand the impact of the system's physical characteristics (orientation, geometry, and morphology). This review of coculture literature delineates the various forms of coculture systems and their respective outcomes when applied to bone tissue engineering. To understand fully the key differences between the different coculture methods, we must appreciate the underlying paradigms of physiological interactions. Recent advances have enabled us to extrapolate these techniques to larger dimensions and higher geometric resolutions. Finally, the contributions of bioreactors, micropatterned biomaterials, and biomaterial interaction platforms are evaluated to give a sense of the sophistication established by a combination of these concepts with coculture systems.

Janardhanan, Sathyanarayana; Wang, Martha O.



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


Human adipose stem cells (hASCs) have been recently used to treat bone defects in clinical practice. Yet there is a need for more optimal scaffolds and cost-effective approaches to induce osteogenic differentiation of hASCs. Therefore, we compared the efficiency of bone morphogenetic proteins (BMP-2 and BMP-7), vascular endothelial growth factor (VEGF), and osteogenic medium (OM) for the osteo-induction of hASCs in 3D culture. In addition, growth factors were tested in combination with OM. Commercially available bioactive glass scaffolds (BioRestore) and biphasic calcium phosphate granules (BoneCeramic) were evaluated as prospective carriers for hASCs. Both biomaterials supported hASC-viability, but BioRestore resulted in higher cell number than BoneCeramic, whereas BoneCeramic supported more significant collagen production. The most efficient osteo-induction was achieved with plain OM, promoting higher alkaline phosphatase activity and collagen production than growth factors. In fact, treatment with BMP-2 or VEGF did not increase osteogenic differentiation or cell number significantly more than maintenance medium with either biomaterial. Moreover, BMP-7 treatment consistently inhibited proliferation and osteogenic differentiation of hASCs. Interestingly, there was no benefit from growth factors added to OM. This is the first study to demonstrate that OM enhances hASC-differentiation towards bone-forming cells significantly more than growth factors in 3D culture. PMID:23361609

Tirkkonen, L; Haimi, S; Huttunen, S; Wolff, J; Pirhonen, E; Sándor, G K; Miettinen, S



Growth characteristics of bone marrow cells from beige mutant, the mouse homologue of the Chediak-Highshi syndrome of man, propagated in semisolid agar cultures  

Microsoft Academic Search

Summary  Suspensions of bone marrow cells from the beige (bg\\/bg) mouse, a homologue of the Chediak-Higashi syndrome (C-HS) of man, and normal mouse bone marrow cells, when stimulated by\\u000a colony-stimulating factor (CSF) from different sources, proliferate in semisolid agar cultures and produce colonies composed\\u000a of granulocytic and\\/or mononuclear cells. Studies with CSF from various sources (embryo and kidney feeder monolayers, conditioned

Harland W. Renshaw; William C. Davis



Acellular bone colonization and aggregate culture conditions diversely influence murine periosteum mesenchymal stem cell differentiation potential in long-term in vitro osteoinductive conditions.  


Periosteum contains mesenchymal stem cells (Pe-MSCs) that contribute to normal bone growth, healing, and turnover; understanding Pe-MSC capabilities may shed light over the treatment of bone defects using tissue engineering. Bone tissue regeneration needs in vitro bone precursors or stem cell coculture onto specific scaffolds but, despite extensive research in the field, very little is known about the matrix structure of the tissue-engineered tissues and the scaffold's effects on cell differentiation. To this purpose we have selected a clonal population (murine Pe-MSCs) that was seeded and differentiated onto an acellular bone scaffold. Cell differentiation was assessed after 3 months and 1 year by molecular, histological, biochemical, and biophysical analyses and results were compared with the same osteoinduced clonal cells cultured as cellular aggregates. Our data show that Pe-MSCs cultured onto acellular bone scaffold develop a complex three-dimensional matrix and an osteoblastic phenotype but do not produce hydroxyapatite (HA); moreover, they seem able to reabsorb the colonized bone scaffold. On the contrary, cells cultured as three-dimensional aggregates differentiate and produce osteoblastic markers and HA nanocrystals. PMID:22494486

Ferro, Federico; Spelat, Renza; D'Aurizio, Federica; Falini, Giuseppe; De Pol, Ilaria; Pandolfi, Maura; Beltrami, Antonio Paolo; Cesselli, Daniela; Beltrami, Carlo Alberto; Curcio, Francesco



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


The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system. PMID:24151432

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



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

PubMed Central

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

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



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

PubMed Central

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

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



Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells.  


Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd. PMID:23225781

Sabatino, Maria Antonietta; Santoro, Rosaria; Gueven, Sinan; Jaquiery, Claude; Wendt, David James; Martin, Ivan; Moretti, Matteo; Barbero, Andrea



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



Clonal succession of hematopoietic cells in long-term bone marrow cultures  

SciTech Connect

The hypothesis that clonal expansion of hematopoietic cells takes place in cultures with succession of functioning clones is tested in this investigation. CBA and CBAT6T6 mice of both sexes aged 8-12 weeks were used. The animals were irradiated with /sup 137/ Cs gamma rays on an IPK apparatus in a dose of 12 Gy. Self-maintenance of a CFU-c was characterized by the number of daughter CFU-c produced by it in irradiated mice during the formation of an 11-day splenic colony. The results provide the authors with an argument in support of the view that CFU-c are incapable of true self-maintenance, but they are members of transient cell populations which mature consecutively, starting from certain earlier clonogenic precursors, or pre-CFU cells.

Gurevich O.A.; Chertkov, I L; Drize, N.I.; Udalov, G.A.



[Differentiation of multipotent mesenchymal stromal cells of bone marrow into cells of cartilage tissue by culturing in three-demential OPLA scaffolds].  


Bone marrow multipotent mesenchymal stromal cells represent a perspective material for engineering of human three-dimensional transplants of cartilage tissue. We are demonstrated the opportunity of the directed differentiation of BM MMSC in cells of cartilage tissue by culturing them in three-dimensional scaffolds, presented by polymer OPLA in medium with inductors of chondrogenesis. For loading cells in porous scaffolds used method which essence consist in saturation of polymeric blocks by cellular suspension with the subsequent centrifugal force of cells in scaffolds and culturing of engineering constructs for 28 days in chondrogenic medium. Histological analysis derived in vitro of three-dimensional transplants showed uniform distribution of cells in the matrix with morphologically distinct chondrocytes-like cells of hyaline cartilage. Immunohistochemical analysis detected aggrecan and collagen type II within the extracellular matrix. Preclinical the researches lead on a livestock of immunodeficient mice have shown not toxicity of the engineering constructs. PMID:17918338

Tepliashin, A S; Korzhikova, S V; Sharifullina, S Z; Rostovskaia, M S; Chupikova, N I; Vasiunina, N Iu; Andronova, N V; Treshchalina, E M; Savchenkova, I P




PubMed Central

Persistent nucleoli were studied in Chinese hamster and human long term cultures, human peripheral blood short term cultures, as well as direct bone marrow preparations. No colchicine or hypotonic treatments were applied and the cells were differentially stained with the Feulgen method and light green. Nucleoli were found to persist in the three systems studied, although to a much greater extent in the long term culture. The persistent nucleolar materials were usually in the form of individualized nucleoli mainly at chromosome ends. They also sometimes existed in a fluidlike or dropletlike condition around the chromosomes. Association of acrocentrics in humans and end-to-end associations in hamsters are likely to result from persistence of nucleoli and the possible effects of colchicine and hypotonic treatments that are usually applied. Other phenomena, such as stickiness at metaphase and separation difficulties and fragmentation at anaphase, may result from persistence of nucleoli. Nucleoli were often associated with large chromosomes and sometimes at sites exhibiting faint or clear constrictions. The possibilities of a partial correspondence between sites of persistence and sites of organization, as well as of the organization of nucleolar materials at sites other than the main organizers, are discussed. The persistent nucleoli were not included in daughter nuclei. They either degenerated in the cytoplasm or were eliminated from the cell. The three systems used may represent different intensities of metabolism reflected in the amounts of nucleolar materials built up and the amount that persists.

Heneen, Waheeb K.; Nichols, Warren W.



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


Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM-MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier-based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM-MSCs in stirred suspension bioreactors using CultiSpher-S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post-inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM-MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10(3) within 30 days, which was superior to the 200-fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM-MSCs in culture, which in turn should facilitate the development of new stem cell-based therapies. Copyright © 2012 John Wiley & Sons, Ltd. PMID:22689330

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



Role of colony-stimulating activity in murine long-term bone marrow cultures: evidence for its production and consumption by the adherent cells  

Microsoft Academic Search

The involvement of colony-stimulating activity (CSA) in murine long-term bone marrow cultures (LTBMC) was studied using bilayer agar cultures. The supernatants of LTBMC were removed. a layer of dense agar was spread over the cells adherent to the bottom of the flask. and fresh myeloid cells were plated as source of CFU-C in an upper agar layer. Large numbers of

Jean-MiChel Heard; Serge Fichelson; Bruno Varet



Bone tissue engineering with human stem cells  

Microsoft Academic Search

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

Darja Marolt; Miomir Knezevic; Gordana Vunjak Novakovic



Three-dimensional culture of mouse bone marrow cells on stroma formed within a porous scaffold: influence of scaffold shape and cryopreservation of the stromal layer on expansion of haematopoietic progenitor cells.  


This study's primary goal was to develop an effective ex vivo expansion method for haematopoietic cells. 3D culture of mouse bone marrow cells was performed in porous scaffolds using a sheet or cube shape. Bone marrow cells were cultured on bone marrow-derived stromal layers formed within the scaffolds and the effect of scaffold shape on the expansion of haematopoietic cells was examined. In some experiments, stromal layers within cubic scaffolds were frozen and then used to culture bone marrow cells after thawing. Results show that after comparison, total cell density and expansion of haematopoietic cells were greater in cultures using the cubic scaffold, suggesting that it was superior to the sheet-like scaffold for expanding haematopoietic cells. When cryopreserved stroma was used, it effectively supported the expansion of haematopoietic cells, and a greater expansion of haematopoietic cells [(erythroid and haematopoietic progenitor cells (HPCs)] was achieved than in cultures with stromal cells that had not been cryopreserved. Expansion of cells using cryopreserved stroma had several other advantages such as a shorter culture period than the conventional method, a stable supply of stromal cells, and ease of handling and scaling up. As a result, this is an attractive method for ex vivo expansion of haematopoietic stem cells (HSCs) and HPCs for clinical use. PMID:22081538

Miyoshi, Hirotoshi; Ohshima, Norio; Sato, Chiaki



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.

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



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

Microsoft Academic Search

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

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



Engraftment defect of cytokine-cultured adult human mobilized CD34(+) cells is related to reduced adhesion to bone marrow niche elements.  


In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short-term homing, leading to loss of cells in non-haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24 h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N-cadherin, osteopontin and vascular cell-adhesion molecule-1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo. PMID:22816563

Kallinikou, Konstantina; Anjos-Afonso, Fernando; Blundell, Michael P; Ings, Stuart J; Watts, Michael J; Thrasher, Adrian J; Linch, David C; Bonnet, Dominique; Yong, Kwee L



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

National Technical Information Service (NTIS)

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

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



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


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

Querido, William; Farina, Marcos



Bone tissue engineering with human stem cells  

PubMed Central

Treatment of extensive bone defects requires autologous bone grafting or implantation of bone substitute materials. An attractive alternative has been to engineer fully viable, biological bone grafts in vitro by culturing osteogenic cells within three-dimensional scaffolds, under conditions supporting bone formation. Such grafts could be used for implantation, but also as physiologically relevant models in basic and translational studies of bone development, disease and drug discovery. A source of human cells that can be derived in large numbers from a small initial harvest and predictably differentiated into bone forming cells is critically important for engineering human bone grafts. We discuss the characteristics and limitations of various types of human embryonic and adult stem cells, and their utility for bone tissue engineering.



A novel ex vivo culture model for inflammatory bone destruction.  


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

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



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


Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes. From a total of 20 × 10(6) BM cells and 120 × 10(6)splenocytes, 3 × 10(6) BM cells along with 20 × 10(6)splenocytes were co-cultured in petri dishes for DC generation; 120 × 10(6) splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. BM cells were the control group cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8?, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA. The number of DCs derived from co-culture of BM and splenocytes (DCs(TME)) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2% and 77.2%, respectively; p<0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCs(TME) compared to BM-derived DCs in the absence of splenocytes (193 pg/ml and 630 pg/ml, respectively; p<0.05). The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCs(TME) could be more advantageous for DC-based immunotherapy and vaccination techniques. PMID:23269574

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



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


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

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



Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures.  

PubMed Central

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage. Images

Borzillo, G V; Sherr, C J



Growth factor-free cultured rat bone marrow derived mesenchymal stem cells towards hepatic progenitor cell differentiation  

Microsoft Academic Search

Induction of mesenchymal stem cells (MSCs) differentiation by growth factors is not likely to be acceptable for clinical application.\\u000a In this study, MSCs and hepatocytes isolated from male Sprague-Dawley rats were indirectly co-cultured by sharing media, without\\u000a any growth factor, for 14 days. Differentiation was investigated by detection of hepatic markers, albumin, ?-fetoprotein (AFP),\\u000a cytokeratin-18 (CK18), and bile ductal epithelial

Tian Zhu Li; Sang-Hyun Shin; Hyun Hee Cho; Jae Hyung Kim; Hwal Suh



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

PubMed Central

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

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



Culture system for bone metabolic studies  

NASA Astrophysics Data System (ADS)

Understanding the mechanisms governing bone remodelling is essential for a clear understanding of not only pathological conditions such as osteoporosis, but also microgravity-induced bone loss. The scope of this MAP project is the further development (including technical development and biological validation) of an ex vivo culture system for trabecular bone explants, which can be submitted to controlled mechanical stimuli. This should allow the evaluation of the causal relationship between biochemical and mechanical parameters and bone remodelling.

Vander Sloten, Jos; Jones, David; Richards, R. Geoff; Vico, Laurence; Gasser, Jürg A.; Koller, Bruno; Pugh, Sydney M.



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


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

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



Association of oxidative stress with postmenopausal osteoporosis and the effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures.  


It has been suggested that oxidative stress is associated with the pathogenesis of osteoporosis. The objective of this study was to explore the association between a marker of oxidative stress and either bone turnover markers or bone mineral density (BMD) in postmenopausal women. In addition, the effects of oxidative stress on the formation of osteoclasts in human bone marrow cell culture were examined. We performed a cross-sectional analysis in healthy postmenopausal women aged 60-78 years (n = 135, 68.2 +/- 4.9). Oxidative stress was evaluated in the serum by measuring 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels. The biochemical markers of bone turnover and areal BMD were measured in all participants. Multivariate linear regression analysis revealed a negative association between 8-OH-dG levels and BMD of the lumbar spine, total hip, femoral neck, and trochanter and positive association with type I collagen C-telopeptide (ICTP) levels. The odds ratio of 8-OH-dG for osteoporosis was 1.54 (1.14-2.31, P = 0.003). In cultures of primary human marrow cells, H2O2 caused concentration-dependent activation of TRAP-positive multinucleated giant cells. H2O2 also increased the area of pits per osteoclast activity assay substrate. RT-PCR showed that H2O2 stimulated the expression of M-CSF and RANKL and increased the RANKL/OPG ratio. The data support the view that oxidative stress is associated with increased bone resorption and low bone mass in otherwise healthy women. In addition, RANKL and M-CSF stimulation induced by oxidative stress may participate in osteoclastogenesis in human bone. PMID:20614110

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



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

Microsoft Academic Search

Although the function and effects of many growth factors and extracellular matrix (ECM) molecules have been described for several periodontal tissues in vivo and in vitro, the molecular interactions involved in the communication between cells of the periodontal ligament and the alveolar bone are poorly understood. To contribute to the identification of such interactions, we have generated co-cultures (CCs) of

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



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


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

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



Selective Expansion of CD34+ Cells from Mouse Bone Marrow Cultured on LH/P MP-Coated Plates with Adequate Cytokines  

PubMed Central

Low-molecular-weight heparin/protamine microparticles (LH/P MPs) serve as carriers for controlled release of heparin-binding cytokines. LH/P MPs were stably coated onto plastic surfaces by drying. The purpose of this study is to evaluate a culture method for selective expansion of CD34+ cells using LH/P MPs as cytokine-binding matrix. Ficoll-purified mouse bone marrow cells (mouse FP-BMCs) containing CD34+ cells were cultured on LH/P MP-coated plates in the presence of stem cell factor (SCF), thrombopoietin (Tpo), and Flt-3 ligand (Flt-3) in hematopoietic progenitor growth medium (HPGM) supplemented with 4% heat-inactivated fetal bovine serum (FBS). After 8 days of culture, the total cell count increased 4.6-fold, and flow cytometry analyses revealed that 23.8% of the initial cells and 57.4% of the expanded cells were CD34 positive. Therefore, CD34+ cells were estimated to have increased 11.0-fold. In contrast, cultured CD34+ cells on uncoated tissue culture plates increased 5.8-fold in an identical medium.

Ishihara, Masayuki; Kanatani, Yasuhiro; Nambu, Masaki; Takikawa, Megumi; Sumi, Yuki; Nakamura, Shingo; Mori, Yasutaka; Hattori, Hidemi; Tanaka, Yoshihiro; Sato, Toshinori



Presence of Mixed Colony-Forming Cells in Long-Term Hamster Bone Marrow Suspension Cultures: Response to Pokeweed Spleen Conditioned Medium  

Microsoft Academic Search

In contrast to the murine system. long-term hamster bone marrow suspension culutres maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen- stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies

Christine E. Eastment; Francis W. Ruscetti; Elizabeth Denholm; Irena Katznelson



Bone Morphogenetic Protein2 Stimulates Differentiation of Cultured Spinal Ligament Cells from Patients with Ossification of the Posterior Longitudinal Ligament  

Microsoft Academic Search

.   Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic bone formation occurring\\u000a in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated\\u000a spinal ligament cells from OPLL patients as well as non-OPLL patients, and established 10 OPLL cell lines and 7 non-OPLL cell\\u000a lines, respectively. We

T. Kon; M. Yamazaki; M. Tagawa; S. Goto; A. Terakado; H. Moriya; S. Fujimura



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

Microsoft Academic Search

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

Mohamadreza Baghaban Eslaminejad; Mahmood Talkhabi; Bahman Zeynali



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

Microsoft Academic Search

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

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



Advances in cell culture  

SciTech Connect

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

Maramorosch, K. (Dept. of Entomology, Rutgers Univ., New Brunswick, NJ (US))



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


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

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



Autologous bone marrow purging with LAK cells.  


In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation. PMID:8013966

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



Bone marrow stem cells.  


The "mesenchymal stem cells (MSCs)" are cells adherent in the bone marrow, which can be isolated to induce differentiation. In contrast to the "embryonic stem cells" whose goal is to develop a new organism, the "MSC adult stem cells" can participate in tissue growth and repair throughout postnatal life. Addition of 5-azacytidine to MSCs in vitro induces the gradual increase in cellular size and begins spontaneous beatings, thereafter differentiating into cardiomyocytes. The "Methods" and "Protocols" to induce structural and functional maturations of MSCs, thus to achieve "Cellular Cardiomyoplasty," are described. With appropriate media, differentiations of MSCs to various kinds of cells such as chondrocytes, osteocytes, and adipocytes are also achievable. PMID:23807784

Duong, Minh Ngoc; Ma, Yu-Ting; Chiu, Ray C J



EP2 and EP4 receptors differentially mediate MAPK pathways underlying anabolic actions of prostaglandin E2 on bone formation in rat calvaria cell cultures.  


Of the four prostaglandin (PG) E receptor subtypes (EP1-EP4), EP2 and EP4 have been proposed to mediate the anabolic action of PGE(2) on bone formation but comparative evaluation studies of EPs on bone formation do not necessarily share a common mechanism, implying that their additional features including downstream MAPK pathways may be beneficial to resolve this issue. We systematically assessed the roles of EPs in the rat calvaria (RC) cell culture model by using four selective EP agonists (EPAs). Consistent with relative expression levels of the respective receptors, multiple phenotypic traits of bone formation in vitro, including proliferation of nodule-associated cells, osteoblast marker expression and mineralized nodule formation were upregulated not only by PGE(2) but equally by EP2A and EP4A, but not by EP1A and EP3A. EP2A and EP4A were effective when cells were treated chronically or pulse-treated during nascent nodule formation. EP2A and EP4A equally stimulated the endogenous PGE(2) production, while EP2A caused a greater increase in cAMP production and c-Fos gene expression compared to EP4A. EP2A and EP4A activated predominantly p38 MAPK and ERK respectively, while c-Jun N-terminal kinase (JNK) was equally activated by both agonists. SB203580 (p38 MAPK inhibitor) blocked the PGE(2) effect on mineralized nodule formation, while U0126 (ERK inhibitor) and dicumarol (JNK inhibitor) were less effective. PGE(2)-dependent phosphorylation of the MAPKs was affected not only by protein kinase (PK)A and PKC inhibitors but also by adenylate cyclase and PKC activators. Co-treatment of RC cells with EP2A or EP4A and bone morphogenetic protein (BMP)2, whose effects on bone nodule formation is known to be, in part, mediated through the PKA and p38 MAPK pathways, resulted in an additive effect on mineralized nodule formation. Further, PGE(2), EP2A and EP4A did not increase BMP2/4 mRNA levels in RC cells, and EP2-induced phosphorylation of p38 MAPK was not eliminated by Noggin. These results suggest that, in the RC cell model, the anabolic actions of PGE(2) on mineralized nodule formation are mediated at least in part by activation of the EP2 and EP4 receptor subtype-specific MAPK pathways, independently of BMP signaling, in cells associated with nascent bone nodules. PMID:19233324

Minamizaki, Tomoko; Yoshiko, Yuji; Kozai, Katsuyuki; Aubin, Jane E; Maeda, Norihiko



Three-dimensional cancer-bone metastasis model using ex-vivo co-cultures of live calvarial bones and cancer cells  

Microsoft Academic Search

One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free-floating live mouse calvarial bone organs in the presence of

Paul Curtin; Helen Youm; Erdjan Salih


Giant Cell Tumor of Bone  


Copyright 2010 American Academy of Orthopaedic Surgeons Giant Cell Tumor of Bone Giant cell tumor of bone (GCT) is a rare, aggressive non-cancerous (benign) tumor. It generally occurs in adults between the ages of ...


Phenomenon of formation of giant fat-containing cells in human bone marrow cultures induced by human serum factor: normal and leukemic patterns.  

PubMed Central

Normal human sera induce the formation of fat-containing cells (FCC) in human bone marrow cultures. A nearly complete monolayer of FCC is formed after 7-14 days of cultivation with 20% human sera in the medium. FCC-inducing activity (FCCIA) is nondialyzable through 14,900-dalton cutoff membrane and is stable at 56 degrees C for 30 min. Abundant FCCIA was found in 83% of normal human sera but in only 20% of sera from untreated patients with different hemopoietic disorders and in 32% of treated leukemic patients. It is suggested that FCCIA may be involved in regulation of the bone marrow microenvironment an that it varies in normal individuals and in patients with different diseases. Images

Svet-Moldavskaya, I A; Zinzar, S N; Svet-Moldavsky, G J; Arlin, Z; Vergara, C; Koziner, B; Clarkson, B D; Holland, J F



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


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

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



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

Microsoft Academic Search

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

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



Signal Transduction in Electrically Stimulated Bone Cells  

Microsoft Academic Search

Background: Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the bio- chemical pathways that are activated in signal transduction when various types of electrical stimulation are ap- plied to bone cells. Methods: Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes



Development of 19F NMR for measurement of [Ca2+]i and [Pb2+]i in cultured osteoblastic bone cells.  


Lead (Pb) has been shown to perturb cellular calcium (Ca) homeostasis, altering sizes and flux rates of cellular pools of exchangeable Ca and impairing Ca-mediated cell processes. To date, however, a direct effect of Pb on intracellular-free Ca2+ has not yet been demonstrated. Heavy metals bind to the commonly used fluorescent Ca ion indicators with greater affinity than does Ca and thereby interfere with the expected Ca-dependent fluorescence. In this study, the fluorinated Ca ion indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA), and 19F NMR were used to measure the free intracellular Ca ion concentration ([Ca2+]i) in the rat osteoblastic bone cell line, ROS 17/2.8. Both Pb and Ca bind to 5F-BAPTA with high affinity, but the Pb-5F-BAPTA comple produces a 19F NMR signal at a chemical shift distinct from 5F-BAPTA and the Ca-5F-BAPTA complex. The apparent dissociation constants for Pb-5F-BAPTA and Ca-5F-BAPTA are 2 X 10(-10) M and 5 X 10(-7) M, respectively, at 30 degrees C, pH 7.1, and Mg2+ (0.5 mM). Thus, this methodology allows for the simultaneous identification and quantification of free Pb and free Ca ion concentrations. Determinations of [Ca2+]i were based on 19F NMR measurements of 5F-BAPTA-loaded ROS 17/2.8 osteoblastic bone cells that were attached to collagen-coated microcarrier beads. Cells were continuously superfused with freshly oxygenated medium at 30 degrees C. Under these conditions, the [Ca2+]i of ROS 17/2.8 cells was observed to be 128 +/- 14 nM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2112459

Schanne, F A; Dowd, T L; Gupta, R K; Rosen, J F



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.



"Bare Bones" Cell Model  

NSDL National Science Digital Library

Using things easily found in the kitchen, learners will identify three parts of a simple cell model. They will see and feel how these parts work together. Educators can prepare gelatin models ahead of time and provide learners with instructions to do the same at home. This activity is featured on pp.11-12 of the "Bones: More Than They Appear" unit of study for 4th, 5th, and 6th grade learners.

Indianapolis, The C.; Creative Street, Inc.



Increased Osteoclast-Like Cells Formation in Long-Term Bone Marrow Cultures from Patients with a Spinal Cord Injury  

Microsoft Academic Search

.   Patients with a spinal cord section loose a significant amount of bone. After paraplegia, bone loss occurs below the lesional\\u000a level and is the more dramatic in iliac bones and in the metaphyseal area of long bones. A peak of urinary calcium and hydroxyprolinuria\\u000a is observed approximately 6 weeks after their lesion. To further understand the mechanisms underlying the

A. Demulder; M. Guns; A. Ismail; E. Wilmet; P. Fondu; P. Bergmann



Effect of bone morphogenetic protein-4 on the expression of Sox2, Oct-4, and c-Myc in human periodontal ligament cells during long-term culture.  


Recent studies demonstrated that the endogenous expression level of Sox2, Oct-4, and c-Myc is correlated with the pluripotency and successful induction of induced pluripotent stem cells. Periodontal ligament cells (PDLCs) have a multilineage differentiation capability and ability to maintain the undifferentiated stage, which makes PDLCs a suitable cell source for tissue repair and regeneration. To elucidate the effect of an in vitro culture condition on the stemness potential of PDLCs, we explored the cell growth, proliferation, cell cycle, and the expression of Sox2, Oct-4, and c-Myc in PDLCs from the passage 1 to 7 with or without the addition of recombinant human bone morphogenetic protein-4 (rhBMP4). Our results revealed that BMP-4 promoted cell growth and proliferation, arrested PDLCs in the S phase of cell cycle, and upregulated the propidium iodinate value. It was revealed that without the addition of rhBMP4, the expression of Sox2, Oct-4, and c-Myc in PDLCs only maintained the nucleus location until passage 3, and then lost the nucleus location subsequently. The mRNA expression in PDLCs further confirmed that the level of Sox2 and Oct-4 peaked at passage 3 and then decreased afterward, whereas c-Myc maintained consistently the upregulation along the passages. After the treatment with rhBMP4, the expression of Sox2, Oct-4, and c-Myc in PDLCs maintained the nucleus location even at passage 7, and the mRNA expression of Sox2 and Oct-4 significantly upregulated at the passages 5 and 7. These results demonstrated that addition of rhBMP-4 in the culture medium could improve the current culture condition for PDLCs to maintain in an undifferentiated stage. PMID:23311397

Liu, Lu; Wei, Xi; Huang, Rong; Ling, Junqi; Wu, Liping; Xiao, Yin



Malignant Tumor Formation after Transplantation of Short-Term Cultured Bone Marrow Mesenchymal Stem Cells in Experimental Myocardial Infarction and Diabetic Neuropathy  

PubMed Central

Rationale Bone marrow (BM)-derived mesenchymal stem cells (MSCs) hold great promise for cardiovascular cell therapy owing to their multipotency and culture-expandability. Objective The aim of the study was to investigate whether MSCs can treat experimental acute myocardial infarction (MI) and diabetic neuropathy. Methods and Results We isolated mononuclear cells from mouse BM and cultured MSCs in a conventional manner. Flow cytometry analyses of these cultured cells at passage four showed expression of typical MSC markers such as CD44 and CD29, but not hematopoietic markers such as c-kit, flk1 and CD34. To determine the therapeutic effects of MSCs, we injected MSCs into the periinfarct area after ligation of the left anterior descending coronary arteries of mice, and as separate experiments injected the same batch of MSCs into hindlimb muscles of mice with diabetic neuropathy. During the follow-up at 4–8 weeks after cell transplantation, growing tumors were observed in 30% of hearts in the MI model, and in 46% of hindlimbs in the diabetic neuropathy model. Histologic examination of the tumors revealed hypercelluarity, pleomorphic nucleoli, cytologic atypia and necrosis, and positive staining for ?-smooth muscle actin, indicative of malignant sarcoma with myogenic differentiation. Chromosomal analysis of these MSCs showed multiple chromosomal aberrations including fusion, fragmentation, and ring formation. Conclusions Genetically unmodified MSCs can undergo chromosomal abnormalities even at early passages and form malignant tumors when transplanted in vivo. These results suggest that careful monitoring of chromosomal status is warranted when in vitro expanded MSCs are used for cell therapy such as for MI.

Jeong, Jin-Ok; Han, Ji Woong; Kim, Jin-Man; Cho, Hyun-Jai; Park, Changwon; Lee, Namho; Kim, Dong-Wook; Yoon, Young-sup



Hypoxic culture and in vivo inflammatory environments affect the assumption of pericyte characteristics by human adipose and bone marrow progenitor cells.  


Previous studies have shown that exposure to a hypoxic in vitro environment increases the secretion of pro-angiogenic growth factors by human adipose-derived stromal cells (hASCs) [Cao Y, et al., Biochem Biophys Res Commun 332: 370-379, 2005; Kokai LE, et al., Plast Reconstr Surg 116: 1453-1460, 2005; Park BS, et al., Biomed Res (Tokyo) 31: 27-34, 2010; Rasmussen JG, et al., Cytotherapy 13: 318-328, 2010; Rehman J, et al., Circulation 109: 1292-1298, 2004]. Previously, it has been demonstrated that hASCs can differentiate into pericytes and promote microvascular stability and maintenance during angiogenesis in vivo (Amos PJ, et al., Stem Cells 26: 2682-2690, 2008; Traktuev DO, et al., Circ Res 102: 77-85, 2008). In this study, we tested the hypotheses that angiogenic induction can be increased and pericyte differentiation decreased by pretreatment of hASCs with hypoxic culture and that hASCs are similar to human bone marrow-derived stromal cells (hBMSCs) in these regards. Our data confirms previous studies showing that hASCs: 1) secrete pro-angiogenic proteins, which are upregulated following culture in hypoxia, and 2) migrate up gradients of PDGF-BB in vitro, while showing for the first time that a rat mesenteric model of angiogenesis induced by 48/80 increases the propensity of both hASCs and hBMSCs to assume perivascular phenotypes following injection. Moreover, culture of both cell types in hypoxia before injection results in a biphasic vascular length density response in this model of inflammation-induced angiogenesis. The effects of hypoxia and inflammation on the phenotype of adult progenitor cells impacts both the therapeutic and the basic science applications of the cell types, as hypoxia and inflammation are common features of natural and pathological vascular compartments in vivo. PMID:21865587

Amos, Peter J; Mulvey, Carolyn L; Seaman, Scott A; Walpole, Joseph; Degen, Katherine E; Shang, Hulan; Katz, Adam J; Peirce, Shayn M



Bone cell interactions through Eph/ephrin  

PubMed Central

Bones cannot properly form or be maintained without cell-cell interactions through ephrin ligands and Eph receptors. Cell culture analysis and evaluation of genetic mouse models and human diseases reveal various ephrins and Eph functions in the skeletal system. Migration, attachment and spreading of mesenchymal stem cells are regulated by ephrinB ligands and EphB receptors. ephrinB1 loss-of-function is associated with craniofrontonasal syndrome (CFNS) in humans and mice. In bone remodeling, ephrinB2 is postulated to act as a “coupling stimulator.” In that case, bidirectional signaling between osteoclastic ephrinB2 and osteoblastic EphB4 suppresses osteoclastic bone resorption and enhances osteoblastic bone formation, facilitating the transition between these two states. Parathyroid hormone (PTH) induces ephrinB2 in osteoblasts and enhances osteoblastic bone formation. In contrast to ephrinB2, ephrinA2 acts as a “coupling inhibitor,” since ephrinA2 reverse signaling into osteoclasts enhances osteoclastogenesis and EphA2 forward signaling into osteoblasts suppresses osteoblastic bone formation and mineralization. Furthermore, ephrins and Ephs likely modulate pathological conditions such as osteoarthritis, rheumatoid arthritis, multiple myeloma and osteosarcoma. This review focuses on ephrin/Eph-mediated cell-cell interactions in bone biology.

Matsuo, Koichi; Otaki, Natsuko



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

Microsoft Academic Search

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis.

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



Donor Age and Mechanosensitivity of Human Bone Cells  

Microsoft Academic Search

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

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



Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro.  


Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli. PMID:20925023

Giovannini, S; Diaz-Romero, J; Aigner, T; Heini, P; Mainil-Varlet, P; Nesic, D



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

PubMed Central

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

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



Differentiation of bone marrow mesenchymal cells to neural cells  

Microsoft Academic Search

Summary  To explore the possibility and condition of differentiation of bone marrow mesenchymal cells (BMSCs) to neural cellsin vitro, BMSCs from whole bone marrow of rats were cultured. The BMSCs of passage 3 were identified with immunocytochemical staining\\u000a of CD44(+), CD71(+) and CD45(?). There were type I and type II cells in BMSCs. Type I BMSCs were spindle-shaped and strong\\u000a positive

Wu Yongchao; Zheng Qixin; Guo Xiaodong; Xie Zhongping; Wang Yuntao; Hao Jie



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

PubMed Central

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



Anti-melanoma vaccinal capacity of CD11c-positive and -negative cell populations present in GM-CSF cultures derived from murine bone marrow precursors.  


We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo. PMID:23146677

Campisano, Sabrina; Mac Keon, Soledad; Gazzaniga, Silvina; Ruiz, María Sol; Traian, Martín Dodes; Mordoh, José; Wainstok, Rosa



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

PubMed Central

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

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



Number of stromal precursors in mouse bone marrow and expression of cytokine genes in primary cultures of mouse bone marrow cells during various periods after immunization with S. typhimurium antigens.  


Administration of S. typhimurium microbial mass to mice was followed by a significant increase (by 3-4 times) in the efficiency of cloning and number of stromal precursors in the femoral bone marrow. These parameters were maximum on days 1-3, but returned to normal by the 8th-15th day after immunization. As differentiated from intact animals, the expression of genes for proinflammatory cytokines IL-1? (day 1 after immunization), IL-6 (days 1-3), TNF-? (days 1, 3, and 6), and IFN-? (days 1-3) was detected in bone marrow cultures from immunized mice. The expression of genes for IFN-?, IL-18, and IFN-? was decreased on days 1, 3, and 6 after immunization of animals, respectively. Gene expression for the anti-inflammatory cytokine IL-4 was observed on day 6 after immunization. Therefore, this system was not characterized by a decrease in the immune response of stromal cells. The stromal component of hemopoietic and lymphoid organs has the vector of influences in response to bacterial antigens. This vector is directed to the stimulation and progression, but not to the suppression of immune reactions. Our results indicate that resident stromal cells play a role in the immune response of the body. PMID:21234434

Gorskaya, U F; Danilova, T A; Mesentzeva, M V; Shapoval, I M; Narovlyansky, A N; Nesterenko, V G



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



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

Microsoft Academic Search

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

Beerelli Seshi; Sanjay Kumar; Debra Sellers



Analysis of bone marrow stem cell.  


In this study we define hematopoietic stem cells (HSCs) as a population of cells that, when sorted as single cells, gives rise to both myeloid as well as lymphoid progeny. We sorted single cells from four populations of CD34+ cells from fetal bone marrow: (1) CD38- HLA-DR-, (2) CD38- HLA-DR+, (3) CD38+ HLA-DR-, and (4) CD38+ HLA-DR+ into liquid culture media supplemented with interleukin-3 (IL-3) IL-6, stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor, erythropoietin, basic fibroblast growth factor (bFGF) and insuline-like growth factor (IGF-1). The HSCs were found in the cell populations lacking CD38, the plating efficiency was highest in the CD34+ CD38- HLA-DR+ cell population (48% n = 12); however, only a small proportion of the CD34+ CD38- HLA-DR+ cells showed both lymphoid and myeloid growth potential. When the identical cell populations were sorted into liquid culture media supplemented with bFGF and IGF-1, cell growth was noted from only 1%-5% of the sorted CD34+ CD38- HLA-DR- cells. The cells have the potential to grow and differentiate in vitro to form complex structures that recapitulate normal bone formation. Serial passages of the progeny from these cultures resulted in the formation of similar structures. PMID:7527679

Terstappen, L W; Huang, S



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


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

Srouji, S; Livne, E



Thyrocalcitonin: Inhibitor of Bone Resorption in Tissue Culture  

Microsoft Academic Search

A partially purified protein from thyroid glands of rats inhibits release of calcium from long bones of embryo rats in tissue culture. Inhibition was more easily detected when resorption of bone was stimulated by parathyroid hormone.

Judith Friedman; Lawrence G. Raisz



Isolation and characterization of rat alveolar bone cells.  


Samples of rat alveolar bone were first treated by collagenase digestion and then used as explants for cell culture. The cells obtained were subcultured and characterized by morphological and functional criteria. Their alkaline phosphatase activity was increased after incubation in 1,25-(OH)2 vitD3 10(-8) M whereas with gingival cells it did not change. The bone derived-cells organized nodular structures, synthesized type I collagen, Gla-protein, few type III collagen, and fibronectin. In the defined culture conditions no mineralization was observed. However, the method used allows to obtain cells from rat alveolar bone displaying some features of the osteoblastic phenotype. PMID:1657392

Bouvier, M; Couble, M L; Hartmann, D J; Magloire, H



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

Microsoft Academic Search

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

E. H. Burger; J. S. van de Gevel; J. C. Gribnau; C. W. Thesingh; R. van Furth



Bone marrow culture in aplastic anemia.  

PubMed Central

Blood and bone marrow granulocyte colony forming units (CFUc) were assayed in 46 patients with aplastic anemia, and the serum was examined for its inhibitory action on normal CFUc growth. All patients showed a gross reduction in colonies and clusters in incidence and absolute number in the bone marrow and blood. Two proliferative abnormalities of CFUc in aplastic anaemia were identified: a significantly higher than normal cluster to colony ratio (P less than 0.05) and a higher than normal ratio of granulocytes to total aggregates in the bone marrow. Eleven out of 34 patients tested had serum inhibitory to normal CFUc. These patients were indistinguishable from the rest on haematological and CFUc culture characteristics, and no correlation between the results of CFUc assay and haematological severity was found. The results suggest that the CFUc is abnormal in aplastic anaemia, the reduction in pool size being related to a failure of self-renewal, but an immunological role in the pathogenesis of aplastic anaemia remains unproven. The close relationship of CFUc incidence to the percentage of granulocyte precursors in the marrow, together with the failure of the CFUc assay to predict clinical severity, limits the practical use of the assay to the confirmation of diagnosis in aplastic anaemia.

Barrett, A J; Faille, A; Balitrand, N; Ketels, F; Najean, Y



The detection of in vivo hematotoxicity of benzene by in vitro liquid bone marrow cultures  

SciTech Connect

Bone marrow toxicity induced by benzene inhalation was investigated in mice using an in vitro bone marrow culture system and a bioassay for leukemia. Donor animals (6-week-old female C57B1/6J) were exposed to 400 ppm benzene 6 hr/day for either 9 days (5 days/week) or for 11 consecutive days. Cell suspensions from long-term bone marrow cultures established from benzene-exposed mice showed a progressive reduction in the number of spleen colony-forming cells (the hematopoietic stem cell) compared to control. Different combinations of adherent cell layers and reinoculated cells showed that cultures containing bone marrow cells from benzene-exposed mice had a lower capacity to maintain stem cell proliferation than normal combinations irrespective of whether benzene-exposed marrow was in the adherent cell layers or reinoculated cells. These results indicate that benzene inhalation produces stem cell injury leading to diminished self-replication and derangement of the adherent marrow population. Bone marrow cultures from benzene-treated mice did not develop detectable transformation in vitro throughout the culture period. Neonatal mice inoculated with cultured cells from benzene-exposed mice have not developed leukemia during an 8-month period after inoculation.

Harigaya, K.; Miller, M.E.; Cronkite, E.P.; Drew, R.T.



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

SciTech Connect

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

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



Cell Culture Made Easy.  

ERIC Educational Resources Information Center

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

Dye, Frank J.



Optimizing stem cell culture  

PubMed Central

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

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



Stem cell culture engineering  

Microsoft Academic Search

Stem cells have the capacity for self renewal and undergo multilineage differentiation. Stem cells isolated from both blastocysts and adult tissues represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self?renewal and differentiation capacity. In generating particular cell types for specific applications,

Gargi Seth; Catherine M. Verfaillie



The secretome of bone marrow mesenchymal stem cells-conditioned media varies with time and drives a distinct effect on mature neurons and glial cells (primary cultures)  

Microsoft Academic Search

Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been\\u000d\\u000a shown to ameliorate the injured central nervous system (CNS). Although\\u000d\\u000a these effects were initially attributed to the putative differentiation\\u000d\\u000a of MSCs towards the neural lineage, it is now known that most of them\\u000d\\u000a are mediated by the secretome. Up to now most in vitro reports have\\u000d\\u000a dealt with the effects

C. A. Ribeiro; A. J. Salgado; J. S. Fraga; N. A. Silva; R. L. Reis; N. Sousa



Plant cell suspension cultures.  


Plant cell suspension cultures are widely used in plant biology as a convenient tool for the investigation of a wide range of phenomena, bypassing the structural complexity of the plant organism in toto. The homogeneity of an in vitro cell population, the large availability of material, the high rate of cell growth and the good reproducibility of conditions make suspension-cultured cells suitable for the analysis of complex physiological processes at the cellular and molecular levels. Moreover, plant cell cultures provide a valuable platform for the production of high-value secondary metabolites and other substances of commercial interest. Here we describe how to initiate and maintain plant cell cultures starting from explants obtained from in vitro germinated seedlings. Isolation of protoplasts from plant cell suspension cultures and regeneration of plants via organogenesis and somatic embryogenesis are also presented. PMID:23073877

Moscatiello, Roberto; Baldan, Barbara; Navazio, Lorella



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

Microsoft Academic Search

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

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



Effect of immunization with polyvinylpyrrolidone on the counts of stromal precursor cells in bone marrow and spleen of CBA and CBA/N mice and cytokine gene expression in primary cultures of these cells.  


Injection of polyvinylpyrrolidone (synthetic type 2 T-independent antigen) stimulated the efficiency of clone-forming efficiency and the content of stromal precursor cells in CBA mice in the femoral bone marrow (almost 3-fold) and in the spleen (by 1.7 times) with the peak within 24 h and normalization by day 3 after immunization. The expression of IL-6, IL-8, and TNF-? genes in bone marrow and spleen cultures from immunized animals appeared on day 1 and disappeared on day 3. Hence, stimulation of stromal tissue in response to polyvinylpyrrolidone immunization was significantly less pronounced in comparison with immunization with S. typhimurium antigens. The counts of stromal precursor cells in these organs did not increase in CBA/N mice not responding to polyvinylpyrrolidone because they had no xid-mutation in Brutton's tyrosine kinase (Btk) gene, and the proinflammatory cytokine genes expression in primary cultures derived from these animals did not increase either. These data indicated that the degree of stromal tissue stimulation in immunized mice correlated with the immune response intensity. This indicated a close relationship between the stromal tissue and immune system. Stromal tissue seemed to be stimulated not only and not so much through the stromal cell Toll-like receptors, but mainly through interactions of immunocompetent and stromal cells, the former presumably playing the leading role in this process. PMID:22808496

Gorskaya, U F; Danilova, T A; Mezentzeva, M V; Shapoval, M M; Nesterenko, V G



Heparin Enhancement of Factors Stimulating Bone Resorption in Tissue Culture  

Microsoft Academic Search

The addition of small amounts of a commercial heparin solution, or a synthetic polysaccharide-sulfuric ester similar in structure to heparin, to the medium in which bone tissue is cultured markedly enhances the amount of bone resorption obtained with suboptimal concentrations of parathyroid extract or other factors which stimulate bone resorption. It is suggested that heparin be considered a \\

Paul Goldhaber



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



Bone-like nodules formed by human bone marrow stromal cells: comparative study and characterization.  


Autologous bone marrow stromal cells have been proposed as an adjuvant in the treatment of bone nonunion. This cell therapy would require the establishment of culture conditions that permit the rapid expansion of these cells ex vivo while retaining their potential for further differentiation. Our aim was to achieve a full differentiation process using human bone marrow aspirates. We first analyzed the effects of mineralization medium (with ascorbic acid and phosphate) and dexamethasone (dex) during the primary culture of human bone marrow stromal (HBMS) cells on the proliferation/differentiation behavior of first-passage cells. The most appropriate schedule was then selected to further characterize this differentiation model. We showed that primary culture of HBMS cells in proliferation medium (DMEM supplemented with 10% fetal calf serum), with a 48-h treatment by mineralization medium and dex resulted in a better osteoblastic differentiation of first-passage cells than primary culture carried out in mineralization medium with or without dex. We showed that culture of HBMS cells under these conditions (primary culture in proliferation medium, followed by subculture in mineralization medium) led to the formation of specifically mineralized bone-like nodules similar to the ones observed with rat bone marrow stromal cells. Our nodules exhibited three distinct cell types, reproducing in vitro a tissue-like structure. This treatment demonstrated an optimal proliferation and expression of osteoblastic markers such as alkaline phosphatase, osteocalcin, and type I collagen. The primary culture allowed the multiplication of the number of adherent progenitor cells at the initial time of plating by a mean factor of 44,000, which was found to be negatively correlated with age. Thus, this differentiation model could provide a new tool to elaborate an autologous cell therapy designed to enhance osteogenesis. PMID:12667552

Schecroun, N; Delloye, C h



Culture of Goblet Cells.  

National Technical Information Service (NTIS)

The invention encompasses isolation, culture and characterization of goblet cells in vitro form mammalian conjuctiva. Goblet cells can be cultured from conjunctiva of such mammals as, e.g., humans, rats, mice, rabbits and the like. In another aspect of th...

D. A. Dartt J. D. Rios M. A. Shatos



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

Microsoft Academic Search

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

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



Stem cells today: B1. Bone marrow stem cells.  


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 on very recent studies. Methods of identifying, culturing, expanding and grafting stem cells are described, including the separation of haemopoietic and mesenchyme cell lines (HSC and MSC) and recent more detailed analyses using numerous CD and other markers to identify very small subsets of stem cells such as multipotent adult progenitor cells (MAPC) and bone marrow stromal stem cells (BMSSC) from MSC. Queries arising on the immense potential of these stem cell lines due to the discovery of epigentic factors and cell fusions influencing their development and potency are described. A section on cord blood stem cells is followed by a detailed discussion on the modern situation regarding the clinical use of stem cells, its recent setbacks due to epigenetic factors, different approaches to the discovery of a highly multipotent bone marrow stem cell, and a brief description of embryological approaches to identifying the basic bone marrow stem cell in very early mammalian embryos. PMID:15588475

Edwards, R G



Giant cell tumor of bone.  


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



Hematopoiesis in the presence of macrophages in long-term bone marrow cultures.  


We have studied the role of macrophages as stromal elements in long-term cultures of murine bone marrow cells. Normal ectopic macrophages were collected from the lung and co-cultured in various ratios with bone marrow cells in Dexter suspension cultures. At weekly intervals, various parameters of the cultures were evaluated and compared with controls. Increased numbers of macrophages in the bone marrow caused a delay in the formation of the confluent stroma, with distinct differences in the pattern of hematopoietic foci in the stroma. Cytologic analysis of nucleated cells in the nonadhering fraction showed that the monocytic macrophages were the eventual line of differentiation in all cultures; however, this phenomenon was accelerated in cultures containing various fractions of macrophages. There was a dose-dependent reduction in the number of myeloid progenitors in the nonadhering and adhering fractions of macrophage-enriched cultures with respect to controls. This effect was mediated through both humoral factors released in the culture and direct interaction between macrophages and bone marrow cells. The result indicate that elevation of the number of macrophages in the bone marrow could result in an impaired process of hematopoiesis. PMID:7656931

Goliaei, B; Soheili, Z; Behboodi, A; Samiei, S



Influence of estrogens on bone resorption in organ culture  

Microsoft Academic Search

Estradiol benzoate, ethinyl estradiol, and human calcitonin were compared for their ability to inhibit the spontaneous or parathyroid extract-induced bone resorption in organ cultures of 19-day fetal rat fibulae. The criteria used for the assessment of bone resorption were: the release of45Ca from paired prelabeled bone rudiments into the culture medium, the dry weight, and the number of osteoclasts per

M. Liskova



Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.  

PubMed Central

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo. Images

Li, Q X; Fan, H



The activin receptor-like kinase 6 Booroola mutation enhances suppressive effects of bone morphogenetic protein 2 (BMP2), BMP4, BMP6 and growth and differentiation factor-9 on FSH release from ovine primary pituitary cell cultures  

Microsoft Academic Search

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused

Julia M Young; Jennifer L Juengel; Kenneth G Dodds; Mhairi Laird; Peter K Dearden; Alan S McNeilly; Kenneth P McNatty; Theresa Wilson



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

Microsoft Academic Search

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

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



Organotypic culture of human bone marrow adipose tissue.  


The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was <0.8 ng/mL under all culture conditions. Dexamethasone promoted adiponectin gene expression, while insulin inhibited it. This finding suggests that dexamethasone, but not insulin, may serve as a powerful adipogenic factor for BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology. PMID:20403027

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



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


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

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



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



Smurf Control in Bone Cells  

PubMed Central

The homologous to the E6-assosiated protein carboxyl terminus (HECT) domain E3 ubiquitin ligase Smurf1 is the first E3 ligase to be implicated in regulating bone cell function. The involvement of Smurf1 in multiple signaling pathways and pathological conditions is presently an area of extensive scientific interest. This review highlights recent works exploring Smurf-regulated biological processes in bone cells and highlights recent discoveries surrounding the regulatory mechanisms modulating its catalytic activity and substrate recognition capability. Moreover, we discuss the relevance of targeting the HECT E3s through the development of small-molecule inhibitors as an anticancer therapeutic strategy.

Xing, Lianping; Zhang, Ming; Chen, Di



Cytochemistry of sheep bone marrow cells  

Microsoft Academic Search

The present study was conducted on bone marrow samples obtained from 15 clinically normal Libyan Barbary sheep. Haemopoietic cells, including those of myelocytic series, erythrocytic series, megakaryocytic series, lymphocytes, plasma cells, monocytes and mitotic cells were identified on the basis of their morphological characteristics in May-Grünwald-Giemsa stained bone marrow smears. Cytochemical reactions of bone marrow cells revealed that all granulocytes,

Mahasen Matug Gawas; Khalid Mohammed Belhaj; AL IZZI


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))



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

Microsoft Academic Search

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

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



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



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


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

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



The anti-inflammatory property of human bone marrow-derived mesenchymal stem/stromal cells is preserved in late-passage cultures.  


Human mesenchymal stem/stromal cells (hMSCs) have been reported to improve neural damage via anti-inflammation and multi-differentiation abilities. Here, we investigated immunosuppression effects of hMSCs by mixed-culturing with interferon-? (IFN?) stimulated BV-2 mouse microglial cells. We show that hMSCs decreased nitrite oxide (NO) production from BV-2 cells in cell density dependent manner. Aged hMSCs and peroxisome proliferator-activated receptor-? (PPAR?) knockdown hMSCs decreased differentiation abilities but maintained NO suppressive function. We finally confirmed NO suppression activities of hMSCs in IFN?-stimulated primary microglia/macrophages. It suggested that hMSCs significantly modified NO production in activated phagocytes and it might be preserved in late passage cultures. PMID:23998421

Song, Dandan; Ohtaki, Hirokazu; Tsumuraya, Tomomi; Miyamoto, Kazuyuki; Shibato, Junko; Rakwal, Randeep; Xu, Zhifang; Hiraizumi, Yutaka; Inoue, Tomio; Shioda, Seiji



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


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



Comparative study of three types of polymer materials co-cultured with bone marrow mesenchymal stem cells for use as a myocardial patch in cardiomyocyte regeneration.  


The purpose of this study was to investigate the most suitable polymer material for supporting stem cell growth as a myocardial patch. After cell isolation and expansion of mouse bone marrow mesenchymal stem cells (BMSC), the cells were induced to differentiate into cardiomyocytes with 5-azacytidine to determine their differentiation potential. BMSCs were also seeded onto three types of polymer material film, including polyurethane (PU), 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)], and polypropylene carbonate (PPC). The results revealed that cell numbers were more abundant on both the PU and P(3HB-co-4HB) material surfaces. Conversely, the surface of PPC was smooth with only cell lysate debris observed. The average cell counts were as follows: 143.78 ± 38.38 (PU group), 159.50 ± 33.07 [P(3HB-co-4HB) group], and 1.40 ± 0.70 (PPC group). There was no statistically significant difference in cell numbers between the PU and P(3HB-co-4HB) groups. A statistically significant difference was identified between the PPC group and both the PU (P1) and P(3HB-co-4HB) groups (P2). Polymer biomaterial patches composed of PU and P(3HB-co-4HB) permit good stem cell growth. P(3HB-co-4HB) has the potential for development as a clinical alternative to current treatment methods for the regeneration of cardiomyocytes in patients with myocardial infarction. PMID:23620011

Niu, Hongxing; Mu, Junsheng; Zhang, Jianqun; Hu, Ping; Bo, Ping; Wang, Yan



Deep wound cultures and bone biopsy in diabetic foot osteomyelitis.  


BACKGROUND: Osteomyelitis is a major complication in patients with diabetic foot ulceration. Accurate pathogenic identification of organisms can aid the clinician to a specific antibiotic therapy thereby preventing the need for amputation. METHODS: All diabetic patients with bone biopsy- confirmed osteomyelitis were included into the study: biopsies were performed either during surgical removal of infected bone, or percutaneously under guided fluoroscopy through non-infected tissue. The depth and extent of the ulcer was assessed using a sterile blunt metal probe. Deep wound cultures were taken from the wound base after sharp debridement. RESULTS: Of 66 cases of suspected Osteomyelitis in 102 joints, 34 patients had both bone biopsies and deep wound cultures over the study period. Thirty two of 34 (94%), had a history of preceding foot ulceration, and in 25 of the cases a positive probe to bone test was recorded. In a high proportion of patients, at least one similar organism was isolated from both the deep wound culture and bone biopsy procedures (n?=?25, 73.5% of cases; p?cultures and bone biopsies, the identical strain was identified in both procedures in a significant proportion of cases (n?=?14, 56% of cases; p?cultures correlate well with osseous cultures and provide a sensitive method in assessing and targeting likely pathogens that cause osseous infections. This will help aid the clinician in guiding antibiotic therapy in centers where bone biopsies may not be readily available. This article is protected by copyright. All rights reserved. PMID:23653368

Malone, M; Bowling, F L; Gannass, A; Jude, E B; Boulton, A J M



Degradation of polysaccharide hydrogels seeded with bone marrow stromal cells.  


In order to produce hydrogel cell culture substrates that are fit for the purpose, it is important that the mechanical properties are well understood not only at the point of cell seeding but throughout the culture period. In this study the change in the mechanical properties of three biopolymer hydrogels alginate, low methoxy pectin and gellan gum have been assessed in cell culture conditions. Samples of the gels were prepared encapsulating rat bone marrow stromal cells which were then cultured in osteogenic media. Acellular samples were also prepared and incubated in standard cell culture media. The rheological properties of the gels were measured over a culture period of 28 days and it was found that the gels degraded at very different rates. The degradation occurred most rapidly in the order alginate > Low methoxy pectin > gellan gum. The ability of each hydrogel to support differentiation of bone marrow stromal cells to osteoblasts was also verified by evidence of mineral deposits in all three of the materials. These results highlight that the mechanical properties of biopolymer hydrogels can vary greatly during in vitro culture, and provide the potential of selecting hydrogel cell culture substrates with mechanical properties that are tissue specific. PMID:21783124

Jahromi, Shiva H; Grover, Liam M; Paxton, Jennifer Z; Smith, Alan M



Cell senescence culturing methods.  


Development of therapeutic approaches that slow or ablate the adverse physiological and pathological changes associated with aging has been considered as an important goal for gerontological research. As cellular senescence is characterized as the basis for aging in organisms, culturing and subculturing of normal human diploid fibroblasts to mimic the in vivo aging processes have been developed as major methods to investigate molecular events involved in aging. It has been established that normal human diploid fibroblasts can proliferate in culture for only finite periods of time. There are many ways to study aging in vitro. In this chapter, we will discuss some of the basic laboratory procedures for cell senescence culturing methods. PMID:23929093

Chen, Huaping; Li, Yuanyuan; Tollefsbol, Trygve O



Perfusion Based Cell Culture Chips  

Microsoft Academic Search

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

A. Heiskanen; J. Emnéus; M. Dufva



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.


Extracellular Matrix-Associated Bone Morphogenetic Proteins Are Essential for Differentiation of Murine Osteoblastic Cells in Vitro  

Microsoft Academic Search

Osteoblastic differentiation is an essential part of bone formation that compensates resorbed bone matrix to maintain its structural integrity. Cells in an osteoblast lineage develop differentiated phe- notypes during a long-term culture in vitro. However, intrinsic mech- anisms whereby these cells differentiate into mature osteoblasts are yet unclear. Bone morphogenetic proteins (BMPs) stimulate osteo- blastic differentiation and bone formation. We




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


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

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



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

SciTech Connect

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

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



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

PubMed Central

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

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



Application of a Novel Widefield Surface Plasmon Resonance Microscope in Cell Imaging and Wound Closure Properties of TGF-?3, BSA\\/HCl and HCl in Cultured Human Bone Cell Monolayer  

Microsoft Academic Search

\\u000a A newly developed Widefield Surface Plasmon Resonance (WSPR) Microscope was used to investigate the morphology of MG63 bone\\u000a cells and their interfacial interactions with ECM proteins. This allowed detailed imaging of cell surface coupling at lateral\\u000a resolution down to ? 500 nm. In this work, bone repair was investigated and modulated by different stimulus including growth\\u000a factors. TGF-?3 is a

Farshid Sefat; Mansour Youseffi; Morgan Denyer


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

Microsoft Academic Search

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity

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



Can bone marrow differentiate into renal cells?  


A considerable plasticity of adult stem cells has been confirmed in a wide variety of tissues. In particular, the pluripotency of bone marrow-derived stem cells may influence the regeneration of injured tissues and may provide novel avenues in regenerative medicine. Bone marrow contains at least hematopoietic and mesenchymal stem cells, and both can differentiate into a wide range of differentiated cells. Side population (SP) cells, which are originally defined in bone marrow cells by high efflux of DNA-binding dye, seem to be a new class of multipotent stem cells. Irrespective of the approach used to obtain stem cells, the fates of marrow-derived cells following bone marrow transplantation can be traced by labeling donor cells with green fluorescence protein or by identifying donor Y chromosome in female recipients. So far, bone marrow-derived cells have been reported to differentiate into renal cells, including mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney, although controversy exists. Further studies are required to address this issue. Cell therapy will be promising when we learn to control stem cells such as bone marrow-derived stem cells, embryonic stem cells, and resident stem cells in the kidney. Identification of factors that support stem cells or promote their differentiation should provide a relevant step towards cell therapy. PMID:12376804

Imai, Enyu; Ito, Takahito



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


Enhancement of ectopic bone formation by bone morphogenetic protein-2 delivery using heparin-conjugated PLGA nanoparticles with transplantation of bone marrow-derived mesenchymal stem cells  

Microsoft Academic Search

This study was performed to determine if a combination of previously undifferentiated bone marrow-derived mesenchymal stem\\u000a cells (BMMSCs) and exogenous bone morphogenetic protein-2 (BMP-2) delivered via heparin-conjugated PLGA nanoparticles (HCPNs)\\u000a would extensively regenerate bone in vivo. In vitro testing found that the HCPNs were able to release BMP-2 over a 2-week\\u000a period. Human BMMSCs cultured in medium containing BMP-2-loaded HCPNs

Sung Eun Kim; Oju Jeon; Jung Bok Lee; Min Soo Bae; Heoung-Jae Chun; Seong-Hwan Moon; Il Keun Kwon



Bone Morphogenetic Protein2 (BMP2) Inhibits Muscle Development and Promotes Cartilage Formation in Chick Limb Bud Cultures  

Microsoft Academic Search

Bone morphogenetic proteins (BMPs) induce ectopic cartilage and bone when implanted intramuscularly in adult rats. Expression data suggest that BMPs signal skeletal development in embryos. An important question is which cells are targets of BMP signaling in adult and embryonic tissues. Here, we examined the effect of BMP-2 on micromass cultures of chick limb bud mesenchyme. We report that BMP-2

D. M. Duprez; M. Coltey; H. Amthor; P. M. Brickell; C. Tickle



Bone development: overview of bone cells and signaling.  


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

Teti, Anna



Structural analysis of proteoglycans synthesized by mineralizing bone cells in vitro in the presence of fluoride  

Microsoft Academic Search

This study investigate the biochemical structure of proteoglycans synthesized during matrix maturation by mineralizing bone cells in vitro, in the presence and absence of fluoride. Bone cells were obtained from rat femur washes and cultured in ? MEM media supplemented with fetal calf serum, ascorbic acid, ?-glycerophosphate and dexamethasone. Cells were characterized as osteoblast-like by the expression of alkaline phosphatase

R. J. Waddington; M. S. Langley



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.  


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



Abnormalities of myeloid progenitor cells after "successful" bone marrow transplantation.  

PubMed Central

We studied recovery of peripheral blood- and bone marrow-derived myeloid progenitor cells (CFU-G,M) in 29 patients who received bone marrow transplants 2 mo to 8.5 yr previously. All patients had normal levels of peripheral blood neutrophils, normal bone marrow cellularity, and a normal myeloid-erythroid ratio. Both peripheral blood- and bone marrow-derived CFU-G,M were markedly reduced compared with normal controls and bone marrow donors [5 +/- 1/10(6) vs. 37 +/- 4/10(6) (P less than 0.001) and 23 +/- 5/2 x 10(5) vs. 170 +/- 21/2 x 10(5) (P less than 0.001)]. Five patients had no detectable CFU-G,M even when 10(6) bone marrow cels were plated. These abnormalities of CFU-G,M were unrelated to age, sex, diagnosis, conditioning regimen, dose of bone marrow cells transplanted, and presence or absence of graft-vs.-host disease. Patients who received either autotransplants or transplants from identical twins also had decreased or absent CFU-G,M indicating that allogeneic factors and posttransplant immune suppressor with methotrexate or corticosteroids were not major determinants of this abnormality. Co-culture of normal or donor peripheral blood or bone marrow mononuclear cells with recipients peripheral blood or bone marrow mononuclear cells, purified T cells, or serum failed to show any evidence of active CFU-G,M suppression. Furthermore, the abnormality of CFU-G,M could not be corrected by the addition of normal syngeneic (donor) hematopoietic cells or serum. Depletion of T-cells from recipient bone marrow by physical techniques resulted in marked increase in CFU-G,M (36 +/- 13 vs. 138 +/- 36; P less than 0.05). The abnormality could be reproduced in vitro by readdition of autologous T cells. In contrast to results with T cell depletion by physical techniques, T cell depletion with a monoclonal anti-T antibody (B7) and complement had no effect. These data indicate that most-transplant recipients have a marked abnormality in CFU-G,M when these cells are cultured in vitro. In at least some of these patients, the decreased cloning efficiency of CFU-G,M appears to be mediated by a suppressive effect of autologous T cells. Images

Li, S; Champlin, R; Fitchen, J H; Gale, R P



Myelo-lymphopoiesis in long-term bone marrow culture  

Microsoft Academic Search

Summary In vitro analysis of early haemopoietic events has been hampered by the absence of culture systems allowing long-term maintainance and proliferation of self-renewing multipotent stem cells. Recently, a liquid culture system has been established which allows in vitro proliferation of haemopoietic stem cells and of haemopoietic precursor and mature end cells for several months. There is good evidence that

H. G. Mergenthaler



Can bone marrow cells give rise to cornea epithelial cells?  


The corneal epithelium plays a critical role in maintaining the cornea's transparency and its avascularity. Severe damage to the limbal region results in serious problems with the corneal surface such as persistent epithelial defects, conjunctivalisation with vascularisation, keratinisation, scarring, etc. with associated profound visual loss. In order to rescue such damaged ocular surfaces, corneal epithelial stem cells were used to reconstruct artificial corneas by employing tissue engineering method. This procedure, however, requires a large limbal graft from the healthy eye and it is not possible in patients who have bilateral lesions. Therefore we should find other autologous cells as a source of cells for the reconstruction of the corneal surface. c-kit+ enriched bone marrow stem cells can give rise to different epithelial cells. So we hypothesize that this might apply to the cornea as well. Cultured cell sheets composed of autologous c-kit+ enriched bone marrow stem cells may be used to reconstruct corneal surfaces and can restore vision in patients with bilateral severe disorders of the ocular surface. PMID:18495366

Liu, Yuan; Wang, Xinwen; Jin, Yan



Cell Culture Models and Methods  

Microsoft Academic Search

\\u000a This chapter reviews methods to harvest and culture several different cell systems commonly employed in cardiac electrophysiology\\u000a research. The cell systems covered fall into three main categories: (1) primary cell culture; (2) cell lines; and (3) stem\\u000a cell-derived cardiomyocytes. Each cell system has its own set of advantages and disadvantages. Often the best choice depends\\u000a on the question an investigator

Alena Nikolskaya; Vinod Sharma


Response Kinetics of Radiation-induced Micronucleated Reticulocytes in Human Bone Marrow Culture  

PubMed Central

The frequency of micronucleated reticulocytes (MN-RETs) in the bone marrow or peripheral blood is a sensitive indicator of cytogenetic damage. While the kinetics of MN-RET induction in rodent models following irradiation have been investigated and reported, information about MN-RET induction of human bone marrow after radiation exposure is sparse. In this report, we describe a human long-term bone marrow culture (LTBMC), established in three-dimensional (3D) bioreactors, which sustains long-term erythropoiesis. Using this system, we measured the kinetics of human bone marrow red blood cell (RBC) and reticulocyte (RET) production, as well as the kinetics of human MN-RET induction following radiation exposure up to 6 Gy. Human bone marrow established in the 3D bioreactor demonstrated an average percentage of RBCs among total viable cells peaking at 21% on day 21. The average percentage of RETs among total viable cells reached a maximum of 11% on day 14, and remained above 5% by day 28, suggesting that terminal erythroid differentiation was still active. Time- and dose-dependent induction of MN-RET by gamma radiation was observed in the human 3D LTBMC, with peak values occurring at approximately 3 days following 1 Gy irradiation. A trend towards delayed peak to 3–5 days post-radiation was observed with radiation doses ? 2 Gy. Our data reveal valuable information on the kinetics of radiation-induced MN-RET of human bone marrow cultured in the 3D bioreactor, a synthetic bioculture system, and suggest that this model may serve as a promising tool for studying MN-RET formation in human bone marrow, thereby providing opportunities to study bone marrow genotoxicity testing, mitigating agent effects, and other conditions that are not ordinarily feasible to experimental manipulation in vivo.

Sun, Hongliang; Tsai, Ying; Nowak, Irena; Dertinger, Stephen D.; David Wu, J. H.; Chen, Yuhchyau



Dynamic single cell culture array  

Microsoft Academic Search

We demonstrate perfusion culture of arrays of individually trapped cells with dynamic microfluidic control of cellular environment, high maintenance of cell isolation, and low cell death. Hydrodynamically trapped cells were shown to adhere and divide at a comparable rate to cells grown randomly on the same glass substrate. This technique will be useful in quantitative single cell studies that require

Dino Di Carlo; Liz Y. Wu; Luke P. Lee



Evaluation of osteogenic cell culture and osteogenic/peripheral blood mononuclear human cell co-culture on modified titanium surfaces.  


This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1?, TGF?1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, 'multi-cell type' interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation. PMID:23531996

Moura, Camilla G; Souza, Maria A; Kohal, Ralf J; Dechichi, Paula; Zanetta-Barbosa, Darceny; Jimbo, Ryo; Teixeira, Cristina C; Teixeira, Hellen S; Tovar, Nick; Coelho, Paulo G



Effect of media perfusion rate on cell seeded 3D bone constructs in vitro  

Microsoft Academic Search

Using a tissue culture system we designed that perfuses culture media through 3D porous cellular constructs, this study tested the effects of media perfusion rate on cell viability, proliferation and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for one week at flow rates of 0.01, 0.1, 0.2 and

B. D. Porter; S. H. Cartmell; R. E. Guldberg



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

PubMed Central

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

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



Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells  

Microsoft Academic Search

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18–29 years, n = 6) and old (aged 68–81 years, n = 5) donors. MSC were serially passaged until reaching maximal life span. Cell growth, markers of cellular senescence, and osteogenic

Karin Stenderup; Jeannette Justesen; Christian Clausen; Moustapha Kassem



Mycobacterium avium complex: significance of isolation from bone marrow culture.  

PubMed Central

Six patients with bone marrow cultures yielding Mycobacterium avium complex were encountered at the Mount Sinai Hospital between 1969 and 1976. One additional isolate of the same mycobacterial species was recovered from splenic cyst fluid of a seventh patient. Because none of the patients had illnesses apparently due to M. avium complex, the isolates were unexpected and unexplained. Six of the seven patients had other acute or chronic infectious processes, occurring alone or superimposed on a preexisting disease. These patients were therefore unusual, because nontuberculous mycobacteria have previously been obtained from bone marrow cultures exclusively in patients who had either disseminated or pleuropulmonary nontuberculous mycobacteriosis. The isolation of M. avium complex from the reticuloendothelial tissue of these seven patients may reflect an asymptomatic infection or alternatively may lack significance. Either premise can only be judged by continued careful evaluation of similar findings.

Kozinn, W P; Damsker, B; Bottone, E J



Giant-cell tumor of bone and aneurysmal bone cyst  

Microsoft Academic Search

A correlated histologic and radiographic study of nine giant-cell tumors, six aneurysmal bone cysts, and one combined lesion is presented. Clinical findings and plain radiographic appearances were found to overlap. Angiographically, the giant-cell tumors were richly vascularized, with a marked intratumoral contrast uptake, occasional irregular tumor vessles, a prominent peritumoral arterial net-work, and early draining veins. Microscopic examination revealed fine,

B. Gunterberg; L.-G. Kindblom; S. Laurin



Biostimulation of bone marrow cells with a diode soft laser.  


In recent years, the use of low-intensity red light in regeneration of soft tissue has been increasingly pursued. As far as hard tissue is concerned, the biostimulating effect of laser has already been demonstrated successfully in more rapid healing of tibial bone fractures in mice at a dosage of 2.4 J. However, the effect of light of a low dose laser directly on osteoblasts has not been investigated yet. The aim of this study was to determine the effect of continuous wave diode laser irradiation on osteoblasts derived mesenchymal cells. Three groups of 10 cultures each were irradiated 3 times (days 3, 5, 7) with a pulsed diode soft laser with a wavelength of 690 nm for 60 s. Another 3 groups of 10 cultures each were used as control groups. A newly developed method employing the fluorescent antibiotic tetracycline was used to compare bone growth on these culture substrates after a period of 8, 12 and 16 days, respectively. It was found that all lased cultures demonstrated significantly more fluorescent bone deposits than the non-lased cultures. The difference was significant, as tested by the Tukey Test (P < 0.0001) in the cultures examined after 16 days. Hence it is concluded that irradiation with a pulsed diode soft laser has a biostimulating effect on osteoblasts in vitro, which might be used in osseointegration of dental implants. PMID:11168247

Dörtbudak, O; Haas, R; Mallath-Pokorny, G



Human progenitor cells for bone engineering applications.  


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

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



Long-Term Self-Maintenance of Hemopoietic Precursors in Bone Marrow Culture Derived from Tumor Necrosis Factor-Deficient Mice Is Not a Result of Neoplastic Transformation  

Microsoft Academic Search

In long-term bone marrow cultures derived from tumor necrosis factor-deficient mice the total cell production and the total duration of hemopoiesis are increased (the latter is comparable with mouse life span). Telomerase activity in cells of nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice increases with time and peaks after 1-year culturing. Karyotyping of nonadherent and

M. A. Ershler; I. N. Nifontova; Yu. V. Ol'shanskaya; T. V. Todriya; I. L. Chertkov; N. I. Drize



Huanglongbing and psyllid cell cultures  

Technology Transfer Automated Retrieval System (TEKTRAN)

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


Cytostatic drug effects on human clonogenic tumor cells and human bone marrow progenitor cells (CFU-C) in vitro  

Microsoft Academic Search

Summary Human tumor cells from a squamous cell carcinoma of the lung and from a malignant melanoma and human bone marrow progenitor cells were cultured in a methylcellulose monolayer system. To obtain tumor material for repeated experiments with human tumor specimens, human xenografts grown in the nude mouse system were used. The cultures were incubated continuously with adriamycin, actinomycin-D, bleomycin,

H. A. Neumann; H. H. Fiebig; R. Engelhardt; G. W. Löhr



Cell cycle phase duration in bone marrow cells from malnourished rats during suckling  

Microsoft Academic Search

The generation time and duration of the different phases of the cell cycle were estimated in cultured bone marrow cells from malnourished and well-nourished rats during the lactation period by the percentage labeled mitosis (PLM) technique after a short pulse treatment with tritiated thymidine. The PLM were obtained in sequential analysis after every 3 h from removing labeled thymidine to

José L. Gómez; Carolina Campos; Pablo Rangel; Rocío Ortiz



BMP-7 in combination with estrogen enhances bone formation in a fracture callus explant culture.  


In postmenopausal women, estrogen withdrawal results in decrease in bone density or osteoporosis. Osteoporosis leads to fracture and retards bone-healing response. Bone morphogenetic protein-7 (BMP-7), a member of the transforming-growth factor-beta superfamily, has been shown as a promising candidate that stimulates bone growth in its application to fracture healing. The purpose of this study was to determine whether BMP-7 could enhance bone formation in the absence of estrogen. Female rats underwent a controlled closed fracture at the midshaft of the right femur. The callus tissues were harvested from the fracture site eight days following the fracture, and were cultured in serum-free media. The explanted callus tissues were then treated with BMP-7, estrogen (E2) or both. We assessed bone formation by measuring alkaline phosphatase (AP) activity, expression of an osteogenic transcription factor, Runt-related transcription factor-2 (Runx2), production of nitric oxide (NO), and calcium mineralization. Supplementation of serum-free cultures with BMP-7 alone increased cell proliferation by twofold, caused a 6.5-fold increase in AP activity, and enhanced calcium mineralization after 48 h. Moreover, BMP-7 in combination with E2 caused a 8.2-fold increase in the AP activity. Runx2 protein expression was increased following stimulation with BMP-7 and E2. Interestingly, E2 induced the amount of NO production by twofold, whereas BMP-7 did not, either alone or with E2. Thus, BMP-7 could enhance early and late markers of bone fracture healing in callus explant cultures, except for NO. BMP-7 could be a promising growth factor in the treatment of fractures as a consequence of osteoporosis. PMID:20453459

Wei, Aiqun; Leong, Anthony; Williams, Lisa; Chung, Sylvia; Shen, Bojiang; Bhargav, Divya; Diwan, Ashish D



Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro  

Microsoft Academic Search

The effect of menaquinone-7 (MK-7; vitamin K2) on oateoclast-like cell formation and osteoclastic bone resorption in rat femoral tissues in vitro was investigated. The bone marrow cells were cultured for 7 days in a a-minimal essential medium (a-MEM) containing a well-known bone resorbing agent [parathyroid hormone (1–34) (PTH) or prostaglandin E2 (PGE2)] with an effective concentration. Osteoclast-like cells were estimated

Masayoshi Yamaguchi; Zhong Jie Ma



Generation of Functional Clonal Cell Lines from Human Bone Marrow Stroma  

Microsoft Academic Search

Five clonal human bone marrow stromal cell lines were isolated from the adherent cell populations in long-term liquid cultures after transfection with the recombinant plasmid pSV3gpt. All the cell-line feeder layers and their conditioned media stimulated the proliferation of committed granulomonocytic stem cells (CFUc) from human bone marrow. The size and number of early erythroid stem cell (BFUe)-derived colonies were

Kenichi Harigaya; Hiroshi Handa



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

PubMed Central

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

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



Epithelial Cells Derived from Swine Bone Marrow Express Stem Cell Markers and Support Influenza Virus Replication In Vitro  

Microsoft Academic Search

The bone marrow contains heterogeneous population of cells that are involved in the regeneration and repair of diseased organs, including the lungs. In this study, we isolated and characterized progenitor epithelial cells from the bone marrow of 4- to 5-week old germ-free pigs. Microscopically, the cultured cells showed epithelial-like morphology. Phenotypically, these cells expressed the stem cell markers octamer-binding transcription

Mahesh Khatri; Yehia M. Saif



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

Microsoft Academic Search

The loss of bone mass observed in aging enhances the risk of fractures. The process of bone repair in aging is slow and limited due to reduced activity of the osteoblasts. Bone marrow stem cells (MSCs) residing in the bone marrow are the progenitors for osteoblasts. The ability to enhance healing of bone defect in aging by MSCs can contribute

S. Srouji; E. Livne



Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells.  


Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10(-10) M with maximum values achieved at 10(-8)M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1 ,25(OH)(2)D(3) during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells. PMID:9173083

Cheng, S L; Zhang, S F; Avioli, L V



Unique and reliable rat model for the assessment of cell therapy: bone union in the rat mandibular symphysis using bone marrow stromal cells.  


Many kinds of bone graft materials have been developed and reported to repair various bone defects. The defects are usually created by surgical resection of pre-existing bone tissue. However, spontaneous healing of bone defects without implantation of materials could be seen, because bone tissue possesses inherent repairing property. The central portion of the lower jaw bone in many animals consists of fibrous tissue and is called the mandibular symphysis. It persists even in old animals and thus can be interpreted as a physiological bone gap or a non-healing bone defect. We implanted calcium phosphate porous ceramics alone or composites of the ceramics and bone marrow stromal cells (BMSCs) into the bone defect (mandibular symphysis) to examine whether it could be filled with new bone tissue, resulting in bone union. Eight weeks after implantation, micro-computed tomography (micro-CT) and histological and biomechanical analyses demonstrated that bone union of the mandibles occurred in all rats with composites but in none of those with ceramics alone. These results showed that the rat mandibular symphysis is a unique bone defect site for the evaluation of bone graft materials. These analyses demonstrated that ceramics alone could not contribute to bone healing in the defect; however, supplementation with BMSCs drastically changed the properties of the ceramics (turning them into osteogenic ceramics), which completely healed the defect. As BMSCs can be culture-expanded using small amounts of bone marrow, the use of the composites might have clinical significance for the reconstruction of various bone tissues, including facial bone. Copyright © 2012 John Wiley & Sons, Ltd. PMID:23255518

Yagyuu, Takahiro; Kirita, Tadaaki; Hattori, Koji; Tadokoro, Mika; Ohgushi, Hajime



A three-dimensional tissue culture model of bone formation utilizing rotational co-culture of human adult osteoblasts and osteoclasts.  


Living bone is a complex, three-dimensional composite material consisting of numerous cell types spatially organized within a mineralized extracellular matrix. To date, mechanistic investigation of the complex cellular level cross-talk between the major bone-forming cells involved in the response of bone to mechanical and biochemical stimuli has been hindered by the lack of a suitable in vitro model that captures the "coupled" nature of this response. Using a novel rotational co-culture approach, we have generated large (>4mm diameter), three-dimensional mineralized tissue constructs from a mixture of normal human primary osteoblast and osteoclast precursor cells without the need for any exogenous osteoconductive scaffolding material that might interfere with such cell-cell interactions. Mature, differentiated bone constructs consist of an outer region inhabited by osteoclasts and osteoblasts and a central region containing osteocytes encased in a self-assembled, porous mineralized extracellular matrix. Bone constructs exhibit morphological, mineral and biochemical features similar to remodeling human trabecular bone, including the expression of mRNA for SOST, BGLAP, ACP5, BMP-2, BMP-4 and BMP-7 within the construct and the secretion of BMP-2 protein into the medium. This "coupled" model of bone formation will allow the future investigation of various stimuli on the process of normal bone formation/remodeling as it relates to the cellular function of osteoblasts, osteoclasts and osteocytes in the generation of human mineralized tissue. PMID:23664885

Clarke, Mark S F; Sundaresan, Alamelu; Vanderburg, Charles R; Banigan, Meredith G; Pellis, Neal R



The animal cell culture collection  

Microsoft Academic Search

Summary  The Animal Cell Culture Collection established by the Advisory Committee and cooperating laboratories at the American Type\\u000a Culture Collection has been described. The description includes procedures and criteria for the acceptance and certification\\u000a of cells, guidelines for future studies, and policies for the selection of cells. The aim of this Collection is to fulfill\\u000a the needs of individual investigators for

C. S. Stulberg; L. L. Coriell; A. J. Kniazeff; J. E. Shannon



Engineering bone tissue substitutes from human induced pluripotent stem cells.  


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

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



Incorporation of bone marrow cells in pancreatic pseudoislets improves posttransplant vascularization and endocrine function.  


Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×10(3) cells. To create bone marrow cell-enriched pseudoislets 2×10(3) islet cells were co-cultured with 2×10(3) bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation. PMID:23875013

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



Analysis of human primary bone cells by fluorescence activated cell scanning: methodological problems and preliminary results  

Microsoft Academic Search

We describe the development of flowcytometrical methods to analyse human primary osteoblast-like cultures obtained from trabecular bone explants in comparison to the human osteosarcoma cell line HOS 58. Two antigens typical of osteoblasts were studied: bone alkaline phosphatase and collagen\\/procollagen I; the non-specific attachment protein fibronectin served as control. The morphology of all different antigens is shown by immunocytochemistry before

Heide Siggelkow; Dirk Hilmes; Katja Rebenstorff; Wiebke Kurre; Iris Engel; Michael Hüfner



Bone Marrow Mesenchymal Stem Cells for Improving Hematopoietic Function: An In Vitro and In Vivo Model. Part 2: Effect on Bone Marrow Microenvironment  

Microsoft Academic Search

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34+ progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were

Soraya Carrancio; Belen Blanco; Carlos Romo; Sandra Muntion; Natalia Lopez-Holgado; Juan F. Blanco; Jesus G. Briñon; Jesus F. San Miguel; Fermin M. Sanchez-Guijo; M. Consuelo Del Cañizo; Pranela Rameshwar



Migration of co-cultured endothelial cells and osteoblasts in composite hydroxyapatite/polylactic acid scaffolds.  


Regeneration of bone in large segmental bone defects requires regeneration of both cortical bone and trabecular bone. A scaffold design consisting of a hydroxyapatite (HA) ring surrounding a polylactic acid (PLA) core simulates the structure of bone and provides an environment for indirect and direct co-culture conditions. In this experiment, human umbilical vein endothelial cells (EC) and normal human primary osteoblasts (OB) were co-cultured to evaluate cell migration and interactions within this biphasic composite scaffold. Both cell types were able to migrate between the different material phases of the scaffold. It was also observed that OB migration increased when they were co-cultured with ECs, whereas EC migration decreased in co-culture. The results show that co-culture of ECs and OBs in this composite biphasic scaffold allows for migration of cells throughout the scaffold and that pre-seeding a scaffold with ECs can increase OB infiltration into desired areas of the scaffold. PMID:21769541

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



[Effect of AMI on proliferative cycle phase of bone marrow cells in mice].  


By using culture of bone marrow cells in vitro and flowcytometry, effect of Astragalus membranaceus injection (AMI) on proliferative cycle phase of bone marrow cells in normal and anemic mice was studied. AMI 40 micrograms/ml (concentration in culture system) can promote normal murine bone marrow cells (BMC) entering proliferative cycle phase (S + M/G2 phase), so do AMI 40 micrograms/ml and AMI 400 micrograms/ml to anemic murine BMC. The results suggested AMI maybe enhance hematopoietic function in mice. PMID:12575040

Zhu, X; Zhu, B



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

SciTech Connect

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

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



Endosperm Cell and Organ Culture  

Microsoft Academic Search

This chapter describes efforts to culture maize endosperm to enable direct developmental and metabolic\\u000a studies of the cereal endosperm organ without the effects of the maternal plant. First, we describe production\\u000a of endosperm in culture through either central cell fertilization in vitro or through isolation of\\u000a embryo sacs following fertilization in vivo. These techniques have been used to culture cereal endosperm\\u000a but

D. Gruis; H. Guo; Q. Tian; O.-A. Olsen


Rat hepatocyte primary cell cultures  

Microsoft Academic Search

Summary  The conditions for obtaining representative, adult rat hepatocyte primary cell cultures were improved such that viable yields\\u000a of 50% of the liver were produced which gave rise to cultures representing 30% of the liver. The survival of the cultures\\u000a in various media was compared revealing that in complex media, particularly containing galactose, survival was improved.

Gary M. Williams; Edilberto Bermudez; Dominick Scaramuzzino



Tendon stem cells: experimental and clinical perspectives in tendon and tendon-bone junction repair  

PubMed Central

Summary Tendon and tendon-bone junction injuries, while heal, have high re-tear rates. Mesenchymal stem cells (MSCs) have great appeal for the promotion of tendon and tendon-bone junction healing because of their high proliferation rate, multi-potency and relative ease of isolation from various tissues. Tendon stem cells have been identified recently and could be an alternative new cell source for tendon and tendon-bone junction repair. In this review, we summarized the in vitro characteristics of tendon stem cells. The evidence supporting the potential use of these cells for tendon and tendon-bone junction repair was presented. In order to therapeutically apply tendon stem cells in the clinical settings, standardization of tendon stem cell culture is essential. Issues relating to the sources, purity, efficacy, safety and delivery of tendon stem cells for tendon and tendon-bone junction repair were summarized and discussed. The direction for future research was suggested.

Lui, Pauline Po Yee; Wong, On Tik



Tendon stem cells: experimental and clinical perspectives in tendon and tendon-bone junction repair.  


Tendon and tendon-bone junction injuries, while heal, have high re-tear rates. Mesenchymal stem cells (MSCs) have great appeal for the promotion of tendon and tendon-bone junction healing because of their high proliferation rate, multi-potency and relative ease of isolation from various tissues. Tendon stem cells have been identified recently and could be an alternative new cell source for tendon and tendon-bone junction repair. In this review, we summarized the in vitro characteristics of tendon stem cells. The evidence supporting the potential use of these cells for tendon and tendon-bone junction repair was presented. In order to therapeutically apply tendon stem cells in the clinical settings, standardization of tendon stem cell culture is essential. Issues relating to the sources, purity, efficacy, safety and delivery of tendon stem cells for tendon and tendon-bone junction repair were summarized and discussed. The direction for future research was suggested. PMID:23738293

Lui, Pauline Po Yee; Wong, On Tik



Short treatment of osteoclasts in bone marrow culture with calcitonin causes prolonged suppression of calcitonin receptor mRNA  

Microsoft Academic Search

Cells exhibiting osteoclast characteristics of calcitonin receptors (CTRs) and tartrate-resistant acid phosphatase (TRAP) histochemistry are formed in murine bone marrow cultures treated with l?,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We have previously demonstrated that CTR mRNA is highly expressed in these cultures. The aim of this study was to investigate homologous regulation of the CTR, and regulation of TRAP expression in osteoclast-like cells

M. Rakopoulos; M. Ikegame; D. M. Findlay; T. J. Martin; J. M. Moseley



Aflatoxins in Mammalian Cell Cultures.  

National Technical Information Service (NTIS)

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

R. A. Chung



Generation of novel bone forming cells (monoosteophils) from LL-37 treated monocytes  

US Patent & Trademark Office Database

In one embodiment, a monocyte derived bone-forming cell population is provided. In one embodiment, the cell population comprises an isolated monocyte cell population treated with an effective dose of LL-37. In another embodiment, a method of producing a population of monocyte-derived bone-forming cells is provided. The method comprises obtaining a blood sample from a subject; isolating a population of monocytes from the blood sample; treating the isolated monocytes with an effective dose of LL-37; and culturing the LL-37 treated monocytes until they differentiate into the population of monocyte-derived bone-forming cells. In another embodiment, a method of treatment for a bone injury or bone disease is provided. The method comprises administering a therapeutically effective amount of a composition to a subject having the bone injury or disease, the composition comprising a population of monoosteophils.



Cell Culture Models for Neurotoxicology  

Microsoft Academic Search

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

Glyn Stacey; Barbara Viviani



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


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. PMID:23523796

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



Bone marrow mesenchymal stem cells: historical overview and concepts.  


This review describes the historical emergence of the concept of bone marrow mesenchymal stem cells (MSCs), summarizing data on Wolf and Trentin's hematopoietic inductive microenvironment; Dexter's hematopoiesis-supportive stromal cells; Friedenstein's osteogenic cells; and Pittenger's trilineal osteoblastic, chondrocytic, and adipocytic precursors; to finally introduce the specific bone marrow mesenchymal stem cells with differentiation potential to four lineages (mesenchymal and vascular smooth muscle lineages), and stromal and immunomodulatory capacities. Two points are the object of detailed discussion. The first point envisions the stem cell attributes (multipotentiality, self-renewal, tissue regeneration, population heterogeneity, plasticity, and lineage priming) compared with that of the paradigmatic hematopoietic stem cell. In the second point, we discuss the possible existence of bone marrow cells with greater differentiation potential, eventually pluripotential cells. The latter point raises the issues of cell fusion, reprogramming, or selection under nonstandardized conditions of rare populations of neuroectodermal origin, or of cells that had undergone mesenchymal-to-epithelial transition. In the last section, we review data on MSC senescence and possible malignant transformation secondary to extensive culture, gene transfer of telomerase, or mutations such as leading to Ewing's sarcoma. The set of data leads to the conclusion that bone marrow MSCs constitute a specific adult tissue stem cell population. The multiple characteristics of this stem cell type account for the versatility of the mechanisms of injured tissue repair. Although MSC administration may be extremely useful in a number of clinical applications, their transplantation is not without risks that must not be overlooked when developing cell therapy protocols. PMID:20565251

Charbord, Pierre



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

PubMed Central

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

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



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

Microsoft Academic Search

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

Ebtehal AL-Rabeah; Hiran Perinpanayagam; Don MacFarland



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

Microsoft Academic Search

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

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



eOSTEO: bone cell function in microgravity assessed in unmanned missions.  

NASA Astrophysics Data System (ADS)

The Canadian OSTEO experiments on board the space shuttle in 1998 gave life scientists their first opportunity to examine bone cell cultures in space Results showed that isolated bone cells in space take longer to regenerate than on Earth This slower regeneration is aggravated by the hastened action of cells that cause bone deterioration The Canadian Space Agency CSA supported Millenium Biologix for the design of the OSTEO mini-lab which tested the growth of cells using a synthetic bone biomaterial Based on the success of the OSTEO mission an automated version of the OSTEO mini-lab eOSTEO has been designed by Millenium Biologix and will be used to perform experimental studies on board of a FOTON recoverable satellite on orbit for 12 days Several Canadian and European projects will allow to assess the influence of microgravity on bone cell survival differentiation metabolism intracellular organization and gene expression The function of various bone cell receptors in the space environment will be tested The automated mini-lab allows stimulating fixing and cooling of bone cells before satellite recovery and media samples can be stored for further analysis Two modules containing four eOSTEO trays will be integrated in the FOTON satellite along with other modules for scientific experiments in life and physical sciences This pioneering study using automated cell culture on board a satellite will provide new technological expertise for space life sciences and produce crucial information on bone cell behavior in the space environment

Cohen, L.; Johnson-Green, P.; Buckley, N.; Dufour, B.; Lefebvre, L.


Calcitonin effects on isolated bone cells  

Microsoft Academic Search

Summary  Calcitonin decreased calcium uptake in specific rat bone cell populations obtained by sequential collagenase digestions of\\u000a calvaria. The calcitonin effect on calcium uptake was observed in the same populations that manifested calcitonin-induced\\u000a increases in cyclic AMP as well as high levels of acid phosphatase and the ability to release45Ca from prelabeled devitalized bone. No effect of calcitonin was observed in

M. Shlossman; M. Brown; E. Shapiro; R. Dziak



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

Microsoft Academic Search

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

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



Cryopreservation of Dedifferentiated Cell Cultures  

Microsoft Academic Search

When Gottlieb Haberlandt made the first efforts to cultivate single isolated plant cells in salt solutions his goal was to\\u000a prove the totipotency of single cells (Haberlandt 1902). The cultivation of isolated plant cells in a chemically defined culture\\u000a medium became possible only after the discovery and application of auxins (Gautheret 1939). Today plant cells as well as tissues\\u000a can

Elke Heine-Dobbernack; Heiko Kiesecker; Heinz Martin Schumacher


The stem cell niches in bone  

PubMed Central

The stem cell niche is composed of a specialized population of cells that plays an essential role in regulating adult stem cell self-renewal and differentiation. In adults, osteoblasts, responsible for osteogenesis, and hematopoietic cells, responsible for hematopoiesis, are closely associated in the bone marrow, suggesting a reciprocal relationship between the two. It was recently discovered that a subset of osteoblasts functions as a key component of the HSC niche (namely, the osteoblastic niche), controlling HSC numbers. HSCs interact not only with osteoblasts but also with other stromal cells, including endothelial cells. Sinusoidal endothelial cells in bone marrow have been revealed as an alternative HSC niche called the vascular niche. In this Review we compare the architecture of these 2 HSC niches in bone marrow. We also highlight the function of osteoblasts in maintaining a quiescent HSC microenvironment and the likely role of the vascular niche in regulating stem cell proliferation, differentiation, and mobilization. In addition, we focus on studies of animal models and in vitro assays that have provided direct insights into the actions of these osteoblastic and vascular niches, revealing central roles for numerous signaling and adhesion molecules. Many of the discoveries described herein may contribute to future clinical treatments for hematopoietic and bone-related disorders, including cancer.

Yin, Tong; Li, Linheng



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.

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



Strain difference of murine bone marrow-derived mast cell functions  

Microsoft Academic Search

Mast cells play an important role for the induction and the expression of allergic re- sponses. In this report, we studied the strain dif- ference of bone marrow-derived murine mast cell (BMMC) functions in vitro. BMMC were induced by in vitro culture of bone marrow cells from BALB\\/c and C57BL\\/6 mice with interleukin (IL)-3 for 4 weeks, stimulated with immunoglobulin

Junko Noguchi; Etsushi Kuroda; Uki Yamashita



Bone marrow aspiration  


... soft tissue inside bones that helps form blood cells. It is found in the hollow part of most bones. Bone marrow aspiration is the removal of a small amount of this tissue in liquid form for examination. See also: Bone marrow biopsy Bone marrow culture


Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro  

Microsoft Academic Search

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is de- scribed. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% auto- logous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to

T. Francey; T. W. Jungi; E. Peterhans



Skeletal Cell Fate Decisions Within Periosteum and Bone Marrow During Bone Regeneration  

PubMed Central

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

Colnot, Celine



Adult stem cells in bone and cartilage tissue engineering.  


The progressive increase in life expectancy within the last century has led to the appearance of novel health related problems, some of those within the musculoskeletal field. Among the latter, one can find diseases such as osteoporosis, rheumatoid arthritis and bone cancer, just to mention some of the most relevant. Other related problems are those that arise from serious injuries, often leading to non-recoverable critical size defects. The therapies currently used to treat this type of diseases/injuries are based on the use of pharmaceutical agents, auto/allotransplant and synthetic materials. However, such solutions present a number of inconveniences and therefore, there is a constant search for novel therapeutic solutions. The appearance of a novel field of science called Tissue engineering brought some hope for the solution of the above mentioned problems. In this field, it is believed that by combining a 3D porous template--scaffold--with an adequate cell population, with osteo or chondrogenic potential, it will be possible to develop bone and cartilage tissue equivalents that when implanted in vivo, could lead to the total regeneration of the affected area. This ideal cell population should have a series of properties, namely a high osteo and chondrogenic potential and at the same time, should be easily expandable and maintained in cultures for long periods of time. Due to its natural and intrinsic properties, stem cells are one of the best available cell types. However, after this sentence, the readers may ask, "Which Stem Cells?". During the last 10/15 years, the scientific community witnessed and reported the appearance of several sources of stem cells with both osteo and chondrogenic potential. Therefore, the present review intends to make an overview of data reported on different sources of adult stem cells (bone marrow, periosteum, adipose tissue, skeletal muscle and umbilical cord) for bone and cartilage regenerative medicine, namely those focusing on the differentiation potential of the latter as well as in vivo proof of concept of their applicability. Simultaneously novel aspects of adult stem cells biotechnology such as their immunogenic characteristics and cell expansion methodologies will also be put forward. The present review also points out on issues such as the bone and cartilage regenerative market, and gives a brief description on bone and cartilage bone biology, so the readers can have a true idea of the current state of the art, and how adult stem cells can be an added value to this field. PMID:18220879

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



Comparison of endometrial regenerative cells and bone marrow stromal cells  

PubMed Central

Background Endometrial regenerative cells (ERC) and bone marrow stromal cells (BMSC) are being used in clinical trials. While they have been reported to have similar characteristics, they have not been directly compared. Methods We compared micro RNA (miRNA) and gene expression profiles, soluble cytokine and growth factor levels and ability to inhibit ongoing mixed leukocyte reaction (MLR) of ERC and BMSC each derived from 6 healthy subjects. Results ERC and BMSC miRNA and gene expression profiles were similar, but not identical; more differences were noted in the expression of genes than in miRNAs. Genes overexpressed in ERCs were more likely to be in immune and inflammation pathways and those overexpressed in BMSCs were more likely to be in stem cell and cancer signaling pathways. In addition, the levels of IL-8 and ICAM-1 were greater in ERC supernatants while the levels of HGF, VEGF, IL-6, CXCL12, TGFB1 and TGFB2 were greater in BMSC supernatants. Additionally, ERC demonstrated greater inhibition of the proliferation of mixed leukocyte cultures. Conclusions These results suggest that the in vivo effects of ERC and BMSC may differ. Multiple properties of stromal cells are responsible for their in vivo effectiveness and ERC may be more effective for some of the clinical applications and BMSC for others. Studies in animal models or clinical trials will be required to more fully characterize the differences between ERC and BMSC.



Generation of natural killer cells from bone marrow precursors in vitro.  

PubMed Central

A simple and highly reproducible bone marrow culture system for the generation of cytolytically active NK cells from immature precursors in the bone marrow is described. The NK cells can be generated with various sources of IL-2, including Con A-conditioned medium, supernatants from IL-2-producing cell lines and recombinant IL-2. Neither IL-1, IL-3 nor alpha/beta interferon induced significant cytotoxicity in bone marrow cells. Identification of the cytotoxic cells as NK cells was based on their phenotypic characteristics (aGM1+, Thy 1 +/-, Ly 1-2- X Ia-, RIL-2 +/-, H2+), as well as their spectrum of target specificity. The deliberate addition of peripheral blood mononuclear cells as a source of mature NK cells and elimination of cells expressing markers specific for mature NK cells indicated that the generated NK cells were descendants of precursors of NK cells harboured in the bone marrow and not derived from mature NK cells contaminating the bone marrow preparations. Thus, it was shown that not only functionally active NK cells but also their precursors are highly dependent on IL-2 for differentiation and growth. This culture system should be helpful in studying the origin of NK cells in relation to other cell lineages as well as the regulation of the maturation of NK cells from their precursors.

Kalland, T



Candidates Cell Sources to Regenerate Alveolar Bone from Oral Tissue  

PubMed Central

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

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



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

PubMed Central

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

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



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


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. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950-1958, 2013. PMID:23868185

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



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


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

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



Identification of a hypoxic population of bone marrow cells  

SciTech Connect

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

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



Renal tubular regeneration by bone marrow—derived cells in a girl after bone marrow transplantation  

Microsoft Academic Search

Recent studies have indicated that bone marrow cells can contribute to regeneration of the kidney in experimental models. However, renal regeneration by apparent bone marrow—derived cells has not been shown previously in humans. The authors here report on a 7-year-old girl who received whole bone marrow transplantation from a male donor, and the contribution of bone marrow cells to the

Masashi Nishida; Hidekazu Kawakatsu; Isao Shiraishi; Shin-ichiro Fujimoto; Takahiro Gotoh; Yoji Urata; Takahiko Ono; Kenji Hamaoka



Bone marrow mesenchymal stem cells can differentiate into type II alveolar epithelial cells in vitro.  


In this study, we demonstrate that BMSCs (bone marrow mesenchymal stem cells) can be successfully differentiated into type II alveolar epithelial cells in vitro under mimic pulmonary microenvironment. BMSCs were co-cultured with MRC-5 cells in modified SAGM (small airway growth medium). The BMSC-derived type II alveolar epithelial cells morphologically resemble human lung epithelial cells. They began to appear after 10 days in co-culture and became morphologically dominant after day 15. Correspondingly, SPC (surfactant protein C), a specific functional marker of human type II alveolar epithelial cells, was detected in differentiated cells by RT-PCR (reverse transcription-PCR) analysis after day 15. Immunostaining analysis revealed the present of scattered SPC-positive cells with a differentiation efficiency of 2.43-4.21%. Our study further showed that the SPC gene expression level in differentiated cells was related to the ratio of BMSCs to MRC-5 cells and the components of modified SAGM. PMID:21542803

Ma, Nan; Gai, Hui; Mei, Ju; Ding, Fang-Bao; Bao, Chun-Rong; Nguyen, David M; Zhong, Hong



Treatment of long tubular bone defect of rabbit using autologous cultured osteoblasts mixed with fibrin  

PubMed Central

The osteogenic potential of autologous cultured osteoblasts mixed with fibrin when transplanted to bone defects was evaluated. Radial shaft defects over 15 mm were made in 30 New Zealand white rabbits. A total of 15 rabbits in the control group underwent an iliac bone graft and 15 rabbits in the experimental group underwent an autologous cultured osteoblast injection mixed with fibrin. Both groups were compared radiologically and 5 rabbits in each group were sacrificed for histological evaluation using H-E and Masson’s trichrome stain at 3, 6, and 9 weeks. Osteogenesis in the control group progressed more rapidly than in the experimental group. However, at 9 weeks, bone formation in both groups were similar and showed no significant difference in terms of the amount of bone formation and the quality of bone union. Autologous cultured osteoblast transplantation mixed with fibrin in bone defects was found to produce bone efficiently.

Jang, Jae-Deog; Lee, Seung-Koo



Cell Electroporation in Bone Tissue  

Microsoft Academic Search

\\u000a Electroporation in the cell membrane occurs following exposure to a high-intensity electric field. Electroporation can be\\u000a used to introduce large molecules into the cell or to induce cell apoptosis by the application of the electric field alone,\\u000a provided that the cell damage is such that it cannot be recovered. Electroporation use in clinical practice is standardized\\u000a in association with drugs,

Milena Fini; Matilde Tschon; Marco Alberghini; Giuseppe Bianchi; Mario Mercuri; Laura Campanacci; Francesco Cavani; Mattia Ronchetti; Francesca de Terlizzi; Ruggero Cadossi


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



Principles of cancer cell culture.  


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



Treatment of Severe Post-traumatic Bone Defects With Autologous Stem Cells Loaded on Allogeneic Scaffolds.  


Mesenchymal stem cells may differentiate into angiogenic and osteoprogenitor cells. the effectiveness of autologous pluripotent mesenchymal cells for treating bone defects has not been investigated in humans. We present a case series to evaluate the rationale of using nucleated cells from autologous bone marrow aspirates in the treatment of severe bone defects that failed to respond to traditional treatments. Ten adult patients (mean age, 49.6-years-old) with severe bone defects were included in this study. lower limb bone defects were ?5 cm3 in size, and upper limb defects ?2 cm.3 before surgery, patients were tested for antibodies to common pathogens. treatment consisted of bone allogeneic scaffold enriched with bone marrow nucleated cells harvested from the iliac crest and concentrated using an fDa-approved device. postsurgery clinical and radiographic follow-up was performed at 1, 3, 6, and 12 months. to assess viability, morphology, and immunophenotype, bone marrow nucleated cells were cultured in vitro, tested for sterility, and assayed for the possible replication of adventitious (contaminating) viruses. In 9 of 10 patients, both clinical and radiographic healing of the bone defect along with bone graft integration were observed (mean time, 5.6 months); one patient failed to respond. no post-operative complications were observed. bone marrow nucleated cells were enriched 4.49-fold by a single concentration step, and these enriched cells were free of microbial contamination. The immunophenotype of adherent cells was compatible with that of mesenchymal stem cells. We detected the replication of Epstein-barr virus in 2/10 bone marrow cell cultures tested. hepatitis b virus, cytomegalovirus, parvovirus b19, and endogenous retrovirus hErV-K replication were not detected. overall, 470 to 1,150 million nucleated cells were grafted into each patient. This case series, with a mean follow-up of almost 2 years, demonstrates that an allogeneic bone scaffold enriched with concentrated autologous bone marrow cells obtained from the iliac crest provides orthopedic surgeons a novel option for treating important bone defects that are unresponsive to traditional therapies. PMID:23065806

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



Interleukin6 Release by Cultured Peripheral Blood Mononuclear Cells Inversely Correlates with Height Velocity, Bone Age, Insulin-like Growth Factor-I, and Insulin-like Growth Factor Binding Protein3 Serum Levels in Children with Perinatal HIV1 Infection  

Microsoft Academic Search

Spontaneous and phytohemagglutinin (PHA)-stimulated interleukin (IL)-6 release by cultured peripheral blood mononuclear cells was related to height velocity, bone age, insulin-like growth factor-I (IGF-I), and IGF binding protein-3 (IGFBP-3) serum level standard deviation scores (SDS) of 32 children [aged 91 (median; range 13–151) months] with human immunodeficiency virus-type 1 (HIV-1) perinatal infection and severe disease. Spontaneous and PHA-stimulated IL-6 release

Maurizio de Martino; Luisa Galli; Francesco Chiarelli; Alberto Verrotti; Maria Elisabetta Rossi; Giuseppe Bindi; Fiorella Galluzzi; Roberto Salti; Alberto Vierucci



Bone Marrow Cells and Myocardial Regeneration  

Microsoft Academic Search

Hematopoietic stem cell (HSC) plasticity and its clinical application have been studied profoundly in the past few years.\\u000a Recent investigations indicate that HSC and other bone marrow stem cells can develop into other tissues. Because of the high\\u000a morbidity and mortality of myocardial infarction and other heart disorders, myocardial regeneration is a good example of the\\u000a clinical application of HSC

Fu-sheng Wang; Cathy Trester



Electromagnetic field change the expression of osteogenesis genes in murine bone marrow mesenchymal stem cells  

Microsoft Academic Search

Summary  In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells (MSCs) stimulated by electromagnetic\\u000a field (EMF) with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of\\u000a cDNA and cRNA.

Dongming Zhao; Hua Wu; Feng Li; Rui Li; Chaoxiong Tao



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

Microsoft Academic Search

Lead (Pb) has been shown to perturb cellular calcium (Ca) homeostasis, altering sizes and flux rates of cellular pools of exchangeable Ca and impairing Ca-mediated cell processes. To date, however, a direct effect of Pb on intracellular-free Ca2+ has not yet been demonstrated. Heavy metals bind to the commonly used fluorescent Ca ion indicators with greater affinity than does Ca

F. A. Schanne; T. L. Dowd; R. K. Gupta; J. F. Rosen



Partial Differential Equation Model of the Bone Remodeling Process: Cell Population Dynamics in Canine Cortical Bone.  

National Technical Information Service (NTIS)

The bone remodeling process in canine cortical bone in the mid-shaft of the humerus is modeled with two forward Kolmogorov equations, convection-diffusion type partial differential equations, representing the osteoclast cells and the osteoblast precursor ...

S. J. Hood



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


Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet-rich plasma (PRP). In this study, we investigated the capability of F/P NP-coated plates to act as a substratum for the proliferation of human adipose-derived stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP-coated plates and grew optimally, with a doubling time of 30 and 32 h in low-concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor-2 (FGF-2) on the F/P NP-coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF-2 on F/P NP-coated plates pretreated with PRP and FGF-2 in a concentration-dependent manner. Thus, F/P NP-coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low-concentration PRP medium supplemented with FGF-2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd. PMID:22473706

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



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

PubMed Central

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

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



Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein*  

PubMed Central

Despite significant progress in our understanding of mesenchymal stem cell (MSC) biology during recent years, much of the information is based on experiments using in vitro culture-selected stromal progenitor cells. Therefore, the natural cellular identity of MSCs remains poorly defined. Numerous studies have reported that CD44 expression is one of the characteristics of MSCs in both humans and mice; however, we here have prospectively isolated bone marrow stromal cell subsets from both human and mouse bone marrow by flow cytometry and characterized them by gene expression analysis and function assays. Our data provide functional and molecular evidence suggesting that primary mesenchymal stem and progenitor cells of bone marrow reside in the CD44? cell fraction in both mice and humans. The finding that these CD44? cells acquire CD44 expression after in vitro culture provides an explanation for the previous misconceptions concerning CD44 expression on MSCs. In addition, the other previous reported MSC markers, including CD73, CD146, CD271, and CD106/VCAM1, are also differentially expressed on those two cell types. Our microarray data revealed a distinct gene expression profile of the freshly isolated CD44? cells and the cultured MSCs generated from these cells. Thus, we conclude that bone marrow MSCs physiologically lack expression of CD44, highlighting the natural phenotype of MSCs and opening new possibilities to prospectively isolate MSCs from the bone marrow.

Qian, Hong; Le Blanc, Katarina; Sigvardsson, Mikael



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



Effect of macrophage migration inhibition factor on the content of stromal precursor cells in mouse bone marrow and efficiency of bone marrow precursor cell cloning in vitro.  


The content of stromal precursor cells in the bone marrow of mice decreased 2-5.7 times 24 h after injection of macrophage migration inhibition factor in doses of 0.1-50 ng/kg, this reduction depending on the dose of inhibition factor. The content of precursor cells in the bone marrow of mice increased 2-fold 24 h after injection of S. typhimurium bacterial mass. One day after injection of S. typhimurium bacterial mass, the count of precursor cells in mouse spleen was 7-fold higher than 24 h after injection of macrophage migration inhibition factor. The efficiency of cloning of mouse bone marrow stromal precursor cells in vitro was suppressed 1.7-2.8 times in the presence of macrophage migration inhibition factor in doses of 0.1 to 50 ng/ml culture medium. The effect of cloning inhibition was preserved, if macrophage migration inhibition factor was added to the culture medium after 2 days of bone marrow cell culturing. In general, macrophage migration inhibition factor inhibits stromal precursor cells in vivo and in vitro. The data also indicate that macrophage migration inhibition factor is not responsible for rapid and sharp increase in the count of stromal precursor cells after immunization of animals. PMID:19513377

Gorskaya, U F; Tretyakov, O U; Suslov, A P; Nesterenko, V G



Bone Repair Cells for Craniofacial Regeneration  

PubMed Central

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

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



Influence of engineered titania nanotubular surfaces on bone cells.  


A goal of current orthopedic biomaterials research is to design implants that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, these implants should result in the formation of a characteristic interfacial layer with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the bone-material interface is needed, as well as the development of new materials and approaches that promote osseointegration. Using anodization, titania interfaces can be fabricated with controlled nanoarchitecture. This study demonstrates the ability of these surfaces to promote osteoblast differentiation and matrix production, and enhance short- and long-term osseointegration in vitro. Titania nanotubular surfaces were fabricated using an anodization technique. Marrow stromal cells (MSCs) were isolated from male Lewis rats and seeded on these surfaces along with control surfaces. The interaction of cells with these surfaces was investigated in terms of their ability to adhere, proliferate and differentiate on them. The experiments were repeated three times with cells from different cultures. All the results were analyzed using analysis of variance (ANOVA). Statistical significance was considered at p<0.05. Furthermore, in vivo biocompatibility was assessed by implanting surfaces subcutaneously in male Lewis rat and performing histological analysis after 4 weeks. Our results indicate that the nanotubular titania surfaces provide a favorable template for the growth and maintenance of bone cells. The cells cultured on nanotubular surfaces showed higher adhesion, proliferation, ALP activity and bone matrix deposition compared to those grown on flat titanium surfaces. In vivo biocompatibility results suggest that nanotubular titania does not cause chronic inflammation or fibrosis. The fabrication routes of titania nano-architectures are flexible and cost-effective, enabling realization of desired platform topologies on existing non-planar orthopedic implants. PMID:17449092

Popat, Ketul C; Leoni, Lara; Grimes, Craig A; Desai, Tejal A



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.



Noninvasive real-time monitoring by alamarblue(®) during in vitro culture of three-dimensional tissue-engineered bone constructs.  


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; Schrooten, Jan



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

PubMed Central

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

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



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

Microsoft Academic Search

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

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



Cartilage tissue engineering by collagen matrix associated bone marrow derived mesenchymal stem cells  

Microsoft Academic Search

In this study we compared the in vitro formation of cartilaginous grafts composed of collagen type I hydrogel with both ovine primary articular chondrocytes (AC) and bone marrow derived mesenchymal stem cells (MSC). During 4 weeks of culture, aggregate properties were quantitatively verified, cell viability and the expression of cartilage markers were assayed. Different microscopic techniques indicated a subdivision of

R. M. Schulz; M. Zscharnack; I. Hanischa; M. Geiling; P. Heppb; A. Bader



Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells  

Microsoft Academic Search

Bone marrow stromal cells (BMSC) can differentiate into various cell types including myocytes, which may be valuable in cellular therapy of myocardial infarction. In an attempt to increase the myogenic commitment of BMSC, we investigated the extent of conversion induced by the demethylation agent 5-azacytidine. BMSC isolated from the adult rat tibia were exposed in culture to 5?M 5-azacytidine for

Alexandrina Burlacu; Ana-Maria Rosca; Horia Maniu; Irina Titorencu; Emanuel Dragan; Victor Jinga; Maya Simionescu



'Pseudomonas aeruginosa' Exotoxin: Effect on Cell Cultures.  

National Technical Information Service (NTIS)

An exotoxin, toxic to both mice and cultured cells, was isolated from cultures of Pseudomonas aeruginosa. Relatively small amounts of the exotoxin inhibited the uptake of uridine and amino acids by Vero cells. Within limits, this toxic action was reversib...

O. R. Pavlovskis F. B. Gordon



Hard tissue formation in a porous HA\\/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow  

Microsoft Academic Search

The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic scaffold material, and then either cultured in

Weibo Zhang; X. Frank Walboomers; Juliette van den Dolder; John A. Jansen



Repair of canine mandibular bone defects with bone marrow stromal cells and porous ?-tricalcium phosphate  

Microsoft Academic Search

Tissue engineering has become a new approach for repairing bone defects. Previous studies have been limited to the use of slow-degradable scaffolds with bone marrow stromal cells (BMSCs) in mandibular reconstruction. In this study, a 30mm long mandibular segmental defect was repaired by engineered bone graft using osteogenically induced autologouse BMSCs seeded on porous ?-tricalcium phosphate (?-TCP, n=5). The repair

Jie Yuan; Lei Cui; Wen Jie Zhang; Wei Liu; Yilin Cao



In vivo alveolar bone regeneration by bone marrow stem cells\\/fibrin glue composition  

Microsoft Academic Search

The repair of alveolar bone defects caused by trauma, periodontal diseases and inflammation is still a challenge for both researchers and clinicians. Although there are many attempts to regenerate bone based on different seed cells and scaffolds, the results are still unsatisfactory. This study aims to clarify whether it could be efficient to reconstruct the alveolar bone by the combination

Liang Zhang; Peihuan Wang; Shenglin Mei; Chenghua Li; Chuan Cai; Yin Ding


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



Nanostructured magnesium increases bone cell density  

NASA Astrophysics Data System (ADS)

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

Weng, Lucy; Webster, Thomas J.



Osteoblast-Prostate Cancer Cell Interaction in Prostate Cancer Bone.  

National Technical Information Service (NTIS)

Prostate cancer cells have a remarkable affinity to develop metastases in bone. Clinical data and laboratory observations both suggest that bone-malignant epithelium interactions play a central role in prostate cancer progression. We have developed an in ...

N. M. Navone



Hydrogel-beta-TCP scaffolds and stem cells for tissue engineering bone.  


Trabecular bone is a material of choice for reconstruction after trauma and tumor resection and for correction of congenital defects. Autologous bone grafts are available in limited shapes and sizes; significant donor site morbidity is another major disadvantage to this approach. To overcome these limitations, we used a tissue engineering approach to create bone replacements in vitro, combining bone-marrow-derived differentiated mesenchymal stem cells (MSCs) suspended in hydrogels and 3-dimensionally printed (3DP) porous scaffolds made of beta-tricalcium-phosphate (beta-TCP). The scaffolds provided support for the formation of bone tissue in collagen I, fibrin, alginate, and pluronic F127 hydrogels during culturing in oscillating and rotating dynamic conditions. Histological evaluation including toluidine blue, alkaline phosphatase, and von Kossa staining was done at 1, 2, 4, and 6 weeks. Radiographic evaluation and high-resolution volumetric CT (VCT) scanning, expression of bone-specific genes and biomechanical compression testing were performed at 6 weeks. Both culture conditions resulted in similar bone tissue formation. Histologically collagen I and fibrin hydrogels specimens had superior bone tissue, although radiopacities were detected only in collagen I samples. VCT scan revealed density values in all but the Pluronic F127 samples, with Houndsfield unit values comparable to native bone in collagen I and fibrin glue samples. Expression of bone-specific genes was significantly higher in the collagen I samples. Pluronic F127 hydrogel did not support formation of bone tissue. All samples cultured in dynamic oscillating conditions had slightly higher mechanical strength than under rotating conditions. Bone tissue can be successfully formed in vitro using constructs comprised of collagen I hydrogel, MSCs, and porous beta-TCP scaffolds. PMID:16376162

Weinand, Christian; Pomerantseva, Irina; Neville, Craig M; Gupta, Rajiv; Weinberg, Eli; Madisch, Ijad; Shapiro, Frederic; Abukawa, Harutsugi; Troulis, Maria J; Vacanti, Joseph P



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

Microsoft Academic Search

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

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



Canine bone marrow cells differentiate into hepatocyte-like cells and placental hydrolysate is a potential inducer  

Microsoft Academic Search

Hepatocyte growth factor (HGF) can stimulate human and rat bone marrow (BM) cells to differentiate into hepatocytes. A human placental hydrolysate (hPH) stimulates proliferation of hepatocytes, but its role as a potential inducer of BM cells to form hepatocytes is unclear. To determine if canine BM cells stimulated with HGF or hPH differentiate into hepatocyte-like cells, BM cells were cultured

Sakurako Neo; Takefumi Ishikawa; Kikumi Ogiwara; Norio Kansaku; Miyuki Nakamura; Masashi Watanabe; Masaharu Hisasue; Ryo Tsuchiya; Takatsugu Yamada



[A comparative study of the dynamics of deep burns healing in using medullary allogenic fibroblast-like mesenchymal stem cells from bone marrow immobilized on biodegrading membranes or taken from cultural plastic].  


Transplantation of suspention of allogenic fibroblast-like mesenchymal stem cells (AFMSC) of the bone marrow and AFMSC immobilized on the biodegradable membrane (BM) stimulates healing of deep burn wounds compared to control (without cell transplantation) because these cells produce biologically active substances into the wound. AFMSC immobilized on BM activate repair processes in the wounds earlier. This may be due to their higher functional activity created in the monolayer by adequate intercellular interactions. To avoid complications caused by low diffuse properties of BM (protein precipitation), BM should be removed from the surface of the burn wound maximum 3 days after its application. PMID:16078651

Rasulov, M F; Sevast'ianov, V I; Egorova, V A; Bogatyrev, S R; Za?denov, V A; Potapov, I V; Onishchenko, N A


Mechanical regulation of bone homeostasis: effects of cyclic pressure on bone cell function in vitro  

Microsoft Academic Search

The present in vitro study exposed rat osteoblasts and osteoclast-precursors in bone marrow cell populations to controlled regimes of cyclic pressure and examined various cell functions that are pertinent to bone homeostasis. The results provided evidence that osteoblasts are sensitive to the frequency of the applied cyclic pressure stimulus. Specifically, compared to controls (static conditions) and cells exposed to cyclic

J. Nagatomi; Bernard P. Arulanandam; Dennis W. Metzger; Alain Meunier; Rena Bizios



Stimulation of bone cell differentiation by low-intensity ultrasound--a histomorphometric in vitro study.  


Several investigations have established a stimulatory effect of low-intensity ultrasound treatment on osteogenesis and fracture healing. The objective of this study was to examine whether the stimulatory effect of low-intensity ultrasound results in increased bone cell activity and/or proliferation. Twenty-four paired triplets of metatarsal bone rudiments of twelve 17-days-old fetal mice were dissected and divided into two groups. One group of bone rudiments was treated with pulsating low-intensity ultrasound (30 mW/cm(2); 1.5 MHz) for 20 min/day for a period of 3 or 6 days. The other group served as controls. After culture, the metatarsal bone rudiments were prepared for computer aided light microscopy. The following histomorphometric parameters were determined: length, width and volume of the calcified cartilage and of the bone collar, and cell number. GLM analysis demonstrated that bone collar volume and calcified cartilage percentage were significantly higher in the ultrasound-stimulated rudiments compared to untreated controls. Further, the calcified cartilage volume bordering the hypertrophic zone was significantly higher than in the center of the bone rudiment. Ultrasound treatment did not change the number of the cells. These results suggest that the stimulatory effect of low-intensity ultrasound on endochondral ossification is likely due to stimulation of bone cell differentiation and calcified matrix production, but not to changed cell proliferation. PMID:15099626

Korstjens, C M; Nolte, P A; Burger, E H; Albers, G H R; Semeins, C M; Aartman, I H A; Goei, S W; Klein-Nulend, J



Effect of Autologous Bone Marrow Stromal Cell Seeding and Bone Morphogenetic Protein-2 Delivery on Ectopic Bone Formation in a Microsphere/Poly(Propylene Fumarate) Composite  

PubMed Central

A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8??g BMP-2?per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2–loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2–impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8??g/mg BMP-2–loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2–impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.

Kempen, Diederik H.R.; Kruyt, Moyo C.; Lu, Lichun; Wilson, Clayton E.; Florschutz, Anthony V.; Yaszemski, Michael J.; Dhert, Wouter J.A.



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

PubMed Central

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

Bodle, Josephine C.; Hanson, Ariel D.



Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.  


The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non-invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo-3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4). PMID:10508913

Walker, L M; Holm, A; Cooling, L; Maxwell, L; Oberg, A; Sundqvist, T; El Haj, A J



Mesenchymal Stem Cells as a Potent Cell Source for Bone Regeneration  

PubMed Central

While small bone defects heal spontaneously, large bone defects need surgical intervention for bone transplantation. Autologous bone grafts are the best and safest strategy for bone repair. An alternative method is to use allogenic bone graft. Both methods have limitations, particularly when bone defects are of a critical size. In these cases, bone constructs created by tissue engineering technologies are of utmost importance. Cells are one main component in the manufacture of bone construct. A few cell types, including embryonic stem cells (ESCs), adult osteoblast, and adult stem cells, can be used for this purpose. Mesenchymal stem cells (MSCs), as adult stem cells, possess characteristics that make them good candidate for bone repair. This paper discusses different aspects of MSCs that render them an appropriate cell type for clinical use to promote bone regeneration.

Zomorodian, Elham; Baghaban Eslaminejad, Mohamadreza



The promotion of the vascularization of decalcified bone matrix in vivo by rabbit bone marrow mononuclear cell-derived endothelial cells  

Microsoft Academic Search

The neovascularization of bone grafting represents an important challenge in bone regeneration. Prevascularization of tissue-engineered bone using endothelial cells (ECs) in vitro sheds light on accelerating the vascularization of bone replacements. In the present study, decalcified bone matrix (DBM) was prevascularized by seeding fibrin gels with ECs that are derived from rabbit bone marrow mononuclear cells (BMMNCs). The compound was

Hongbo Tan; Bin Yang; Xiaojun Duan; Fuyou Wang; Ying Zhang; Xuhong Jin; Gang Dai; Liu Yang



Treatment of Chronic Wounds With Bone Marrow-Derived Cells  

Microsoft Academic Search

Background:Recentevidenceindicatesthatbonemar- row contains stem cells with the potential for differen- tiation into a variety of tissues, including endothelium, liver, muscle, bone, and skin. It may thus be plausible that bone marrow-derived cells can provide progenitor and\\/or stem cells to wounds during healing. Our objec- tive in this study was to establish proof of principle that bone marrow-derived cells applied to

Evangelos V. Badiavas; Vincent Falanga



A microfluidic system for automatic cell culture  

NASA Astrophysics Data System (ADS)

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

Huang, Chun-Wei; Lee, Gwo-Bin



VEGF and bone cell signalling: an essential vessel for communication?  


Vascular endothelial growth factor (VEGF) is an endothelial cell survival factor and is required for effective coupling of angiogenesis and osteogenesis. Although central to bone homeostasis, repair and the pathobiology that affect these processes, the precise mechanisms coupling endothelial cell function within bone formation and remodelling remain unclarified. This review will (i) focus on the potential directionality of VEGF signalling in adult bone by identifying the predominant source of VEGF within the bone microenvironment, (ii) will summarize current VEGF receptor expression studies by bone cells and (iii) will provide evidence for a role for VEGF signalling during postnatal repair and osteoporosis. A means of understanding the directionality of VEGF signalling in adult bone would allow us to most effectively target angiogenic pathways in diseases characterized by changes in bone remodelling rates and enhance bone repair when compromised. PMID:23129289

Clarkin, Claire E; Gerstenfeld, Louis C



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


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

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



Ultrastructure of cultured cells from Schistosoma japonicum  

Microsoft Academic Search

Ultrastructures and their dynamic changes of the cultured cells from Schistosoma japonicum were observed in the present experiments. Several types—including polygonal, round granular, deltaic fan-shaped and flagellated cells—were found in the cultures. The polygonal cells took a major ratio in the cultures from adult S. japonicum, while the majority from schistosomula was round granular cells. The ultrastuctures on the cell

Hui-Fen Dong; Xiao-Bei Chen; Zhen-Ping Ming; Qin-Ping Zhong; Ming-Sen Jiang



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.



Establishment of primary cell cultures: Experiences with 155 cell strains  

Microsoft Academic Search

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

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



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.

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



Effects of inhibition of microtubule assembly on bone mineral release and enzyme release by human breast cancer cells.  


When supernates from the established human breast cancer cell line MCF-7 were applied to fetal rat long bones that had been labeled with 45Ca and devitalized to remove endogenous bone cells, mineral was released from the bones. The release of bone mineral by MCF-7 supernates was associated with increased basal release of hydrolytic enzyme activity by the tumor cells. The basal release of lysosomal enzymes and collagenolytic activity by MCF-7 cells with approximately twice that of mouse 3T3 cells, which did not cause mineral release by the fetal rat bones. Release of hydrolytic enzymes and bone mineral-releasing activity was increased by colchicine and vinblastine, drugs that inhibit microtubule assembly, but not affected by lumicolchicine. Time-course experiments performed on MCF-7 cells with or without colchicine showed that release of cathepsin D and collagenolytic activity was associated more closely with release of bone mineral and degradation of bone matrix than was the release of N-acetylglucosaminidase. The release of previously incorporated [3H]proline from the bones exposed to MCF-7 cell cultures was more closely associated with release of collagenolytic activity by MCF-7 cells than with release of cathepsin D or N-acetylglucosaminidase. These data suggest that breast cancer-mediated bone resorption in vitro is positively correlated with release of hydrolytic enzymes by the tumor cells, and release of these enzymes is enhanced by disassembly of microtubules. PMID:6256415

Eilon, G; Mundy, G R



Clinical use of bone marrow, bone marrow concentrate, and expanded bone marrow mesenchymal stem cells in cartilage disease.  


Mesenchymal stem cells (MSCs) from bone marrow (BM) are widely used for bone and less for cartilage tissue regeneration due to their self-renewal and differentiating properties into osteogenic or chondrogenic lineages. This review considers the last decade of clinical trials involving a two-step procedure, by expanding in vitro MSCs from BM, or the so called "one-step" procedure, using BM in toto or BM concentrate, for the regeneration of cartilage and osteochondral tissue defects. The following conclusions were drawn: (1) Cartilage defects that can be repaired by the two-step technique are about twice the size as those where the one-step method is used; (2) the two-step procedure is especially used for the treatment of osteoarthritic lesions, whereas the one-step procedure is used for osteochondral defects; (3) the number of transplanted cells ranges between 3.8×10(6) and 11.2×10(6) cells/mL, and the period of cell culture expansion of implanted MSCs varies widely with regard to the two-step procedure; (4) hyaluronic or collagenic scaffolds are used in all the clinical studies analyzed for both techniques; (5) the follow-up of the two-step procedure is longer than that of the one-step method, despite having a lower number of patients; and, finally, (6) the mean age of the patients (about 39 years old) is similar in both procedures. Clinical results underline the safety and good and encouraging outcomes for the use of MSCs in clinics. Although more standardized procedures are required, the length of follow-up and the number of patients observed should be augmented, and the design of trials should be implemented to achieve evidence-based results. PMID:23030230

Veronesi, Francesca; Giavaresi, Gianluca; Tschon, Matilde; Borsari, Veronica; Nicoli Aldini, Nicolò; Fini, Milena



Growth inhibition of marek's disease T?lymphoblastoid cell lines by chicken bone?marrow?derived macrophages activated in vitro  

Microsoft Academic Search

Studies have been performed on the induction of cytostatic activity of cultured chicken bone?marrow?derived macrophages. Cultured macrophages were exposed to supernatants of ConA?stimulated spleen cell cultures (lymphokines), lipopolysaccharide (LPS), ConA or infectious bursal disease virus (IBDV). Cytostatic activity of macrophages was examined by testing their growth?inhibiting effect on T?lymphoblastoid Marek's disease cell lines RP?1, HP?2 and MSB?1 as target cells.

V. Von Bülow; A. Klasen



Mesenchymal stem cells from patients to assay bone graft substitutes.  


Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg(2+) ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg(2+) and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg(2+) apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase. PMID:23129455

Manfrini, M; Di Bona, C; Canella, A; Lucarelli, E; Pellati, A; D'Agostino, A; Barbanti-Bròdano, G; Tognon, M



Treatment of long tubular bone defect of rabbit using autologous cultured osteoblasts mixed with fibrin  

Microsoft Academic Search

The osteogenic potential of autologous cultured osteoblasts mixed with fibrin when transplanted to bone defects was evaluated.\\u000a Radial shaft defects over 15 mm were made in 30 New Zealand white rabbits. A total of 15 rabbits in the control group underwent\\u000a an iliac bone graft and 15 rabbits in the experimental group underwent an autologous cultured osteoblast injection mixed with\\u000a fibrin.

Seok-Jung Kim; Jae-Deog Jang; Seung-Koo Lee



Intra-bone marrow-bone marrow transplantation facilitates hemopoietic recovery including dendritic cells  

Microsoft Academic Search

In this report, we provide evidence using a serial bone marrow transplantation (BMT) protocol that intra-bone marrow (IBM)-BMT (IBM-BMT) can efficiently reconstitute the hemopoietic system with cells of donor origin, in contrast to conventional intravenous (IV)-BMT (IV-BMT). Furthermore, the hematolymphoid system of secondary recipients that had received bone marrow cells (BMCs) from primary recipients treated with IBM-BMT recovered earlier than

Susumu Baba; Muneo Inaba; Hiroshi Iwai; Mitsuru Taira; Keizo Takada; Hiroko Hisha; Toshio Yamashita; Susumu Ikehara



A 3D in vitro bone organ model using human progenitor cells.  


Three-dimensional (3D) organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF) of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0±0.3%), pre/osteoclastic (14.0±1.4%) and mesenchymal/osteoblastic (44.0±8.4%) phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides), resorption (by N-terminus collagen-I telopeptides and phosphate levels) and osteoclastic activity (by TRAP-5b) only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants. PMID:21604244

Papadimitropoulos, Adam; Scherberich, Arnaud; Güven, Sinan; Theilgaard, Naseem; Crooijmans, Hendrikus J A; Santini, Francesco; Scheffler, Klaus; Zallone, Alberta; Martin, Ivan



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



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.



Bone Morphogenetic Protein 4 Mediates Human Embryonic Germ Cell Derivation  

PubMed Central

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and/or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)–polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

Hiller, Marc; Liu, Cyndi; Blumenthal, Paul D.; Gearhart, John D.



Preparation of primary cell cultures from lamprey  

Microsoft Academic Search

The lamprey is an important model for studies of evolution and comparative biology. The ability to culture cells from lamprey tissues makes it possible to employ an in vitro approach to address basic questions in these areas. Methods are described for the initiation of cell cultures derived from tissues of adult and larval sea lamprey (Petromyzon marinus). Primary cultures initiated

Chunguang Ma; Paul Collodi



Bone marrow necrosis - initial presentation in sickle cell anemia  

PubMed Central

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

Shafiq, Maria; Ali, Natasha



Engineering bone tissue from human embryonic stem cells  

PubMed Central

In extensive bone defects, tissue damage and hypoxia lead to cell death, resulting in slow and incomplete healing. Human embryonic stem cells (hESC) can give rise to all specialized lineages found in healthy bone and are therefore uniquely suited to aid regeneration of damaged bone. We show that the cultivation of hESC-derived mesenchymal progenitors on 3D osteoconductive scaffolds in bioreactors with medium perfusion leads to the formation of large and compact bone constructs. Notably, the implantation of engineered bone in immunodeficient mice for 8 wk resulted in the maintenance and maturation of bone matrix, without the formation of teratomas that is consistently observed when undifferentiated hESCs are implanted, alone or in bone scaffolds. Our study provides a proof of principle that tissue-engineering protocols can be successfully applied to hESC progenitors to grow bone grafts for use in basic and translational studies.

Marolt, Darja; Campos, Ivan Marcos; Bhumiratana, Sarindr; Koren, Ana; Petridis, Petros; Zhang, Geping; Spitalnik, Patrice F.; Grayson, Warren L.; Vunjak-Novakovic, Gordana



Marrow stromal fibroblastic cell cultivation in vitro on decellularized bone marrow extracellular matrix.  


The in vitro biocompatibility of decellularized bone marrow extracellular matrix was evaluated. Following a freeze-thaw cycle, sectioned discs of fresh frozen rat metaphyseal bone were sequentially incubated in solutions of hypertonic, then hypotonic Ringer's solution, followed by deoxycholic acid, then DNAase I. The adequacy of decellularization of marrow stroma was examined by light microscopy. Marrow stromal fibroblastic cells were harvested by dispersion of rat long bone marrow, followed by concentration by discontinuous Ficoll-Paque gradient centrifugation. The fibroblastic cells were expanded by in vitro cultivation, and second passage cells were cryopreserved until needed. Cryopreserved marrow stromal cells were applied dropwise to sections of decellularized bone marrow extracellular matrix, and cultured in BJGb medium with 20% fetal bovine serum for ten days. Mature cultures were formalin fixed, decalcified, and embedded in paraffin. Light microscopy of hematoxylin and eosin stained sections showed individual spindle cells invading the upper portion of the decellularized extracellular matrix, and also a monolayer of spindle cells on the upper surfaces of exposed trabecular and cortical bone. This experiment showed that decellularized marrow extracellular matrix is a biocompatible three dimensional in vitro substrate for marrow stromal fibroblastic cells. PMID:19778536

Dutra, Timothy F; French, Samuel W



PTHrP increases RANKL expression by stromal cells from giant cell tumor of bone.  


Giant cell tumor of bone (GCT) presents with numerous osteoclast-like multinucleated giant cells that are principally responsible for the extensive bone resorption by the tumor. Although the precise etiology of GCT remains uncertain, the accumulation of giant cells is partially due to the high expression of the receptor activator of nuclear factor-?B ligand (RANKL) from the neoplastic stromal cells. Here, we have investigated whether parathyroid hormone-related protein (PTHrP) plays a role in the pathogenesis of GCT. Immunohistochemistry results revealed PTHrP expression in the stromal cells of the tumor, and that its receptor, the parathyroid hormone type 1 receptor (PTH1R), is expressed by both the stromal cells and giant cells. PCR and Western blot analyses confirmed the expression of PTHrP and PTH1R by isolated stromal cells from five patients presenting with GCT. Treatment of GCT stromal cells with varying concentrations of PTHrP (1-34) significantly increased both RANKL gene expression and the number of multinucleated cells formed from RAW 264.7 cells in co-culture experiments, whereas inhibition of PTHrP with a neutralizing antibody decreased RANKL gene expression. These results suggest that PTHrP is expressed within GCT by the stromal cells and can contribute to the abundant RANKL expression and giant cell formation within the tumor. PMID:22102368

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



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.



Osteogenic matrix sheet-cell transplantation using osteoblastic cell sheet resulted in bone formation without scaffold at an ectopic site.  


We previously reported that in vivo bone formation could be observed in composites of porous hydroxyapatite (HA) scaffolds and cultured mesenchymal stem cells (MSCs). In the present study, we developed a new method for transplantation of cultured MSCs without the necessity of using a scaffold to form bone tissue. MSCs were culture-expanded and lifted as cell sheet structures. These cell sheets, designated osteogenic matrix sheets, showed positive alkaline phosphatase (ALP) staining, high ALP activities and high osteocalcin (OC) contents, indicating their osteogenic potential. We transplanted these sheets into subcutaneous sites in rats to assess whether they possessed in vivo bone-forming capability. The transplanted sheets showed mineralized matrix together with osteocytes and an active osteoblast lining, indicating new bone formation, at 6 weeks after transplantation. HA scaffolds were also wrapped with the sheets to make HA/sheet composites and implanted into subcutaneous sites in rats. Histological sections of the composites revealed bone formation in the HA pores at 4 weeks after implantation. Our present results indicate that MSCs can be cultured as sheet structures, and the resulting sheets themselves or HA-sheet composites represent osteogenic implants that can be used for hard tissue reconstruction. PMID:18493911

Akahane, Manabu; Nakamura, Akifumi; Ohgushi, Hajime; Shigematsu, Hideki; Dohi, Yoshiko; Takakura, Yoshinori



Response of bone and cartilage cells to biomaterials in vivo and in vitro.  


In vivo and in vitro models have been developed to study the bone/material interface. The in vivo model exploits the osteogenesis that accompanies marrow ablation of the rat tibia and uses morphological and biochemical changes in extracellular organelles, called matrix vesicles, as markers of the healing process. Matrix vesicles, which are associated with primary bone formation and calcification, are produced by osteoblasts and are sensitive to cellular and environmental regulation. In bone adjacent to bone-bonding implants, matrix vesicle number increases, as does its alkaline phosphatase activity. In bone adjacent to nonbonding materials, matrix vesicle activity is inhibited. The materials exert systemic effects which can also be studied by use of matrix vesicles. Cell models are needed in order for the specificity of the cellular response to the material to be understood. By the use of culture plates sputter-coated with implant materials, the response of cells can be studied under controlled conditions. Comparison of the response of costochondral chondrocytes at two stages of endochondral development demonstrates that the effects of various materials are surface- and cell-maturation-dependent. Cells cultured on Ti exhibited increased alkaline-phosphatase-specific activity, whereas those cultured on Al2O3 have decreased enzyme activity. PMID:8246298

Boyan, B D; Schwartz, Z; Hambleton, J C



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



Interleukin-6 in the bone marrow microenvironment promotes the growth and survival of neuroblastoma cells  

PubMed Central

Neuroblastoma, the second most common solid tumor in children, frequently metastasizes to the bone marrow and the bone. Neuroblastoma cells present in the bone marrow stimulate the expression of interleukin-6 (IL-6) by bone marrow stromal cells (BMSC) to activate osteoclasts. Here we have examined whether stromal-derived IL-6 also has a paracrine effect on neuroblastoma cells. An analysis of the expression of IL-6 and its receptor IL-6R in 11 neuroblastoma cell lines indicated the expression of IL-6 in 7 cell lines and of IL-6R in 9 cell lines. Treatment of IL-6R positive cells with rhIL-6 resulted in STAT-3 and Erk 1/2 activation. Culturing IL-6R positive neuroblastoma cells in the presence of BMSC or rhIL-6 increased proliferation and protected tumor cells from etoposide-induced apoptosis, whereas it had no effect on IL-6R negative tumor cells. In vivo, neuroblastoma tumors grew faster in the presence of a paracrine source of IL-6. IL-6 induced the expression of cyclooxygenase-2 in neuroblastoma cells with concomitant release of prostaglandin-E2, that increased the expression of IL-6 by BMSC. Supporting a role for stromal-derived IL-6 in patients with neuroblastoma bone metastasis, we observed elevated levels of IL-6 in the serum and bone marrow of 16 patients with neuroblastoma bone metastasis, and in BMSC derived from these patients. Altogether the data indicate that stromal-derived IL-6 contributes to the formation of a bone marrow microenvironment favorable to the progression of metastatic neuroblastoma.

Ara, Tasnim; Song, Liping; Shimada, Hiroyuki; Keshelava, Nino; Russell, Heidi V.; Metelitsa, Leonid S.; Groshen, Susan G.; Seeger, Robert C.; DeClerck, Yves A.



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



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

PubMed Central

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

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.



The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells  

PubMed Central

Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells.

Tirkkonen, Laura; Halonen, Heidi; Hyttinen, Jari; Kuokkanen, Hannu; Sievanen, Harri; Koivisto, Anna-Maija; Mannerstrom, Bettina; Sandor, George K. B.; Suuronen, Riitta; Miettinen, Susanna; Haimi, Suvi



The effects of vibration loading on adipose stem cell number, viability and differentiation towards bone-forming cells.  


Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d(-1) for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells. PMID:21613288

Tirkkonen, Laura; Halonen, Heidi; Hyttinen, Jari; Kuokkanen, Hannu; Sievänen, Harri; Koivisto, Anna-Maija; Mannerström, Bettina; Sándor, George K B; Suuronen, Riitta; Miettinen, Susanna; Haimi, Suvi



Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells  

SciTech Connect

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

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



Bone cell responses of titanium blasted with bioactive glass particles.  


Surface modification of Ti-based metals is an important issue in improving the bone cell responses and bone-implant integration. Blasting Ti with granules (mostly alumina) is commonly used to prepare a clean surface and provide a level of roughness. In this study, glass granules with a bioactive composition were used as the blasting source to improve the surface bioactivity and biocompatibility of a Ti substrate. Bioactive glass particles with a composition of 70SiO(2) * 25CaO * 5P(2)O(5) were prepared using a sol-gel method. A Ti disc was blasted with glass particles using a dental blasting unit (BG-Ti). A Ti disc blasted with commercial spherical-shaped glass (G-Ti) and a disc without blasting (Ti) were also prepared for comparison. The blasted Ti contained a large number of glass particles after the blasting process. The surface roughness of the samples in ascending order was G-Ti>BG-Ti>Ti. Murine-derived preosteoblasts (MC3T3-E1) were seeded on the samples, and the cell growth, differentiation, and mineralization behaviors were observed. The osteoblastic cells attached well and spread actively over all the sample groups with extensive cytoskeletal processes. The level of cell growth on the BG-Ti showed a continual increase with culturing up to 7 days, showing good cell viability. However, there was no significant difference (ANOVA, p<0.05) with respect to the G-Ti and Ti groups. In particular, the alkaline phosphatase (AP) activity of the cells was significantly higher on the BG-Ti than on the other groups after culturing for 14 days. Moreover, the mineralization behavior of the cells, as assessed by Alizarin S Red, was superior on the BG-Ti to that observed on the other groups after culturing for 14 and 28 days. Overall, the blasting of Ti with a bioactive glass composition is considered beneficial for producing substrates with enhanced osteogenic potential. PMID:19737811

Choi, Chang-Rak; Yu, Hye-Sun; Kim, Chul-Hwan; Lee, Jae-Hoon; Oh, Chung-Hun; Kim, Hae-Won; Lee, Hae-Hyoung



A thixotropic nanocomposite gel for three-dimensional cell culture  

NASA Astrophysics Data System (ADS)

Thixotropic materials, which become less viscous under stress and return to their original state when stress is removed, have been used to deliver gel-cell constructs and therapeutic agents. Here we show that a polymer-silica nanocomposite thixotropic gel can be used as a three-dimensional cell culture material. The gel liquefies when vortexed-allowing cells and biological components to be added-and resolidifies to trap the components when the shear force from spinning is removed. Good permeability of nutrients and gases through the gel allows various cell types to proliferate and be viable for up to three weeks. Human mesenchymal stem cells cultured in stiffer gels developed bone-like behaviour, showing that the rheological properties of the gel can control cell differentiation. No enzymatic, chemical, or photo-crosslinking, changes in ionic strength or temperature are required to form or liquefy the gel, offering a way to sub-culture cells without using trypsin-a protease commonly used in traditional cell culture techniques.

Pek, Y. Shona; Wan, Andrew C. A.; Shekaran, Asha; Zhuo, Lang; Ying, Jackie Y.



Clonal Analysis of Bone Marrow and Macrophage Cultures.  

National Technical Information Service (NTIS)

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. (ERA c...

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



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.



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

Microsoft Academic Search

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

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



Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates.  


Human-induced pluripotent stem cells (hiPSCs) may represent an ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem cells using a synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xenogeneic-free culture medium. Here, we tested the hypothesis that iPSCs could be maintained in an undifferentiated state in this xeno-free culture system and subsequently be differentiated into mesenchymal stem cells (iPS-MSCs). hiPSCs were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD90+,CD73+, CD31-, CD34-, and CD45-) and their ability to differentiate in vitro into adipogenic, chondrogenic, and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immunocompromised mice for 8 weeks. MicroCT and histologic analyses demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies confirmed the participation of the transplanted hiPS-MSCs in the regenerated bone. These results demonstrate that hiPSCs cultured in a xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo. PMID:22415987

Villa-Diaz, L G; Brown, S E; Liu, Y; Ross, A M; Lahann, J; Parent, J M; Krebsbach, P H



Cell Sources for Bone Tissue Engineering: Insights from Basic Science  

PubMed Central

One of the goals of bone tissue engineering is to design delivery methods for skeletal stem/progenitor cells to repair or replace bone. Although the materials used to retain cells play a central role in the quality of the constructs, the source of cells is key for bone regeneration. Bone marrow is the most common cell source, but other tissues are now being explored, such as the periosteum, fat, muscle, cord blood, and embryonic or induced pluripotent stem cells. The therapeutic effect of exogenous stem/progenitor cells is accepted, yet their contribution to bone repair is not well defined. The in vitro osteo- and/or chondrogenic potential of these skeletal progenitors do not necessarily predict their differentiation potential in vivo and their function may be affected by their ability to home correctly to bone. This review provides an overview of animal models used to test the efficacy of cell-based approaches. We examine the mechanisms of endogenous cell recruitment during bone repair and compare the role of local versus systemic cell recruitment. We discuss how the normal repair process can help define efficacious cell sources for bone tissue engineering and improve their methods of delivery.



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.



Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization.  


Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5 x 10(6) cells/cm(2) and upon confluency were reseeded as either low density (50 cells/cm(2)) or high density (8 x 10(4) cells/cm(2)) cultures. The cells were expanded through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic morphology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothelial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system. PMID:19452230

Eslaminejad, Mohamadreza Baghaban; Nadri, Samad



Bone marrow as a potential source of hepatic oval cells.  


Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability. PMID:10325227

Petersen, B E; Bowen, W C; Patrene, K D; Mars, W M; Sullivan, A K; Murase, N; Boggs, S S; Greenberger, J S; Goff, J P



Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films  

Microsoft Academic Search

In this study, the adhesion, proliferation and morphology of rabbit anterior cruciate ligament (ACL) cells and bone marrow stromal cells (bMSCs) on synthetic biodegradable polymeric films were investigated. Tissue culture polystyrene (TCP) was used as control. Seven biodegradable polymers were used; they are as follows: poly(?-caprolactone) (PCL), poly(dl-lactide) (d-PLA), poly(l-lactide) (l-PLA), PLA\\/PCL (50:50), PLA\\/PCL (75:25), high molecular weight (HMW) poly(dl-lactide–co-glycolide

H. W. Ouyang; J. C. H. Goh; X. M. Mo; S. H. Teoh; E. H. Lee



Cell-based approaches to the engineering of vascularized bone tissue.  


This review summarizes recent efforts to create vascularized bone tissue in vitro and in vivo through the use of 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 union or non-union. Treatment approaches with the use of 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 two-dimensional and three-dimensional 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



Red cell salvage and reinfusion in pediatric bone marrow donors  

Microsoft Academic Search

We evaluated the use of a semi-automated processing technique to salvage red blood cells from pediatric bone marrow donors to minimize the risk of severe anemia following bone marrow harvest and ABO incompatibility in the recipient. Sixty healthy, HLA-matched, pediatric donors of bone marrow hematopoietic cells with a median age 8.0 years (2–19) were studied. Thirteen of the donor–recipient pairs

M Kletzel; M Olszewski; K Danner-Koptik; K Coyne; PR Haut



Arthroplasty of the lunate using bone marrow mesenchymal stromal cells  

Microsoft Academic Search

Mesenchymal stromal cells have the potential to differentiate into a variety of mesenchymal tissues such as bone, cartilage\\u000a and ligaments. The potential for the regeneration of bone with cartilage coverage has still not been achieved. We evaluated\\u000a the ability of bone marrow mesenchymal stromal cells to regenerate osteochondral defects in the cavity of the lunate in an\\u000a animal model. Autologous

Arne Berner; Carola Pfaller; Thomas Dienstknecht; Johannes Zellner; Michael Müller; Lukas Prantl; Richard Kujat; Carsten Englert; Bernd Fuechtmeier; Michael Nerlich; Peter Angele



[Osteoregulation by Physiological Stresses. Stress and cell communication between bone cells].  


Bone is constantly renewed by the balanced action of bone formation and bone resorption both of which mainly occur at the bone surface. This restructuring process called "bone remodeling" is important not only for normal bone mass and strength, but also for mineral homeostasis. Bone remodeling is stringently regulated by communication between bone component cells such as osteoclasts, osteoblasts and osteocytes. An imbalance of this process is often linked to various bone diseases. During bone remodeling, resorption by osteoclasts precedes bone formation by osteoblasts. Based on the osteocyte location within the bone matrix and the cellular morphology, it is proposed that osteocytes potentially contribute to the controls of bone remodeling as well as sensing mechanical stress. PMID:24162599

Nakashima, Tomoki



Isolation and Characterization of Reticuloendotheliosis Virus Transformed Bone Marrow Cells  

Microsoft Academic Search

Summary Transformed cells have been isolated from the bone marrow of reticuloendotheliosis virus (REV)-infected, moribund chicks. These cells have been maintained in vitro through more than 30 serial passages. The cells induce solid tumors when inoculated into the wing web of day-old chickens, and cell-free filtrates induce reticuloendotheliosis. Virus particles recovered after cocultivation of bone marrow cells and chicken embryo

Ray B. Franklin; Reynaldo L. Maldonado; Henry R. Bose



Heparanase inhibits osteoblastogenesis and shifts bone marrow progenitor cell fate in myeloma bone disease.  


A major cause of morbidity in patients with multiple myeloma is the development and progression of bone disease. Myeloma bone disease is characterized by rampant osteolysis in the presence of absent or diminished bone formation. Heparanase, an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate chains, is upregulated in a variety of human cancers including multiple myeloma. We and others have shown that heparanase enhances osteoclastogenesis and bone loss. However, increased osteolysis is only one element of the spectrum of myeloma bone disease. In the present study, we hypothesized that heparanase would also affect mesenchymal cells in the bone microenvironment and investigated the effect of heparanase on the differentiation of osteoblast/stromal lineage cells. Using a combination of molecular, biochemical, cellular and in vivo approaches, we demonstrated that heparanase significantly inhibited osteoblast differentiation and mineralization, and reduced bone formation in vivo. In addition, heparanase shifts the differentiation potential of osteoblast progenitors from osteoblastogenesis to adipogenesis. Mechanistically, this shift in cell fate is due, at least in part, to heparanase-enhanced production and secretion of the Wnt signaling pathway inhibitor DKK1 by both osteoblast progenitors and myeloma cells. Collectively, these data provide important new insights into the role of heparanase in all aspects of myeloma bone disease and strongly support the use of heparanase inhibitors in the treatment of multiple myeloma. PMID:23895995

Ruan, Jian; Trotter, Timothy N; Nan, Li; Luo, Rongcheng; Javed, Amjad; Sanderson, Ralph D; Suva, Larry J; Yang, Yang



Mechanism by which MLO-A5 Late Osteoblasts\\/Early Osteocytes Mineralize in Culture: Similarities with Mineralization of Lamellar Bone  

Microsoft Academic Search

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly\\u000a elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone\\u000a type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity\\u000a increase with time, features of

C. Barragan-Adjemian; D. Nicolella; V. Dusevich; M. R. Dallas; J. D. Eick; L. F. Bonewald



Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.  


In this study, we have observed dental pulp stem cells (SBP-DPSCs) performances on different scaffolds, such as PLGA 85:15, hydroxyapatite chips (HA) and titanium. Stem cells were challenged with each engineered surface, either in plane cultures or in a rotating apparatus, for a month. Gingival fibroblasts were used as controls. Results showed that stem cells exerted a different response, depending on the different type of textured surface: in fact, microconcavities significantly affected SBP-DPSC differentiation into osteoblasts, both temporally and quantitatively, with respect to the other textured surfaces. Actually, stem cells challenged with concave surfaces differentiated quicker and showed nuclear polarity, an index of secretion, cellular activity and matrix formation. Moreover, bone-specific proteins were significantly expressed and the obtained bone tissue was of significant thickness. Thus, cells cultured on the concave textured surface had better cell-scaffold interactions and were induced to secrete factors that, due to their autocrine effects, quickly lead to osteodifferentiation, bone tissue formation, and vascularization. The worst cell performance was obtained using convex surfaces, due to the scarce cell proliferation on to the scaffold and the poor matrix secretion. In conclusion, this study stresses that for a suitable and successful bone tissue reconstruction the surface texture is of paramount importance. PMID:17565721

Graziano, Antonio; d'Aquino, Riccardo; Cusella-De Angelis, Maria Gabriella; De Francesco, Francesco; Giordano, Antonio; Laino, Gregorio; Piattelli, Adriano; Traini, Tonino; De Rosa, Alfredo; Papaccio, Gianpaolo



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

Microsoft Academic Search

The goal of this work was to develop and validate a whole bone organ culture model to be utilized in biomimetic mechanotransduction\\u000a research. Femurs harvested from 2-day-old neonatal rat pups were maintained in culture for 1 week post-harvest and assessed\\u000a for growth and viability. For stimulation studies, femurs were physiologically stimulated for 350 cycles 24 h post-harvest\\u000a then maintained in culture for 1 week

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



Intra-bone marrow bone marrow transplantation rejuvenates the B-cell lineage in aged mice  

Microsoft Academic Search

Age-related reductions in the frequency and absolute number of early B lineage precursors in the bone marrow of aged mice have been reported. Reversal of B-cell lineage senescence has not been achieved. Age-related impairment of the B-cell lineage is caused by the decreasing functionality of hematopoietic and B lineage precursors, and reduced efficacy of bone marrow stromal cells that constitute

Daisuke Hida; Naoki Ishiguro; Masataka Haneda; Yoshiyuki Ishida; Haruhiko Suzuki; Ken-ichi Isobe; K-i Isobe



Gene and Protein Expression During Differentiation and Matrix Mineralization in a Chondrocyte Cell Culture System  

Microsoft Academic Search

.   Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation\\u000a of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by\\u000a enzymatic digestion from rat fetuses were seeded at a final density of 105 cell\\/cm2 and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf

N. Kergosien; J.-M. Sautier; N. Forest



Bones of contention: Marrow-derived cells in myocardial regeneration  

Microsoft Academic Search

Almost 7 years have passed since the initial publication reporting that bone marrow cells regenerate infarcted myocardium. The subsequent years produced hundreds of investigations that ran the gamut of findings from validation to disproof. Undeterred by the concurrent debate, clinical trials ensued to test the safety and efficacy of bone marrow-derived cell population for autologous therapy in clinical treatment of myocardial

Mark A. Sussman; Charles E. Murry



Migration of bone marrow stem cells in ischaemic brain  

Microsoft Academic Search

Stem cell therapy has been demonstrated to be effective in the management of haematological malignancy and solid cancer, but its role in neurodegenerative conditions remains uncertain. We hypothesize that: (1) ventricular delivery of bone marrow stem cells improves functional outcome in experimental ischaemia of the mouse brain; and (2) this improved outcome is due to migration of bone marrow stem

W. S. Poon; G. Lu; K. S. Tsang; X. L. Zhu; G. G. Chen; H. K. Ng


Esophageal stem cells and 3D-cell culture models.  


The following on esophageal stem cells and 3D-cell culture models contains commentaries on metaplasia through transdifferentiation and through stem cells; transcription factors that may determine an intestinal-like phenotype; the?in vitro, organotypic cell culture models; and the role of stem cells in Barrett's esophagus and its dysplastic progression. PMID:21950821

Souza, Rhonda F; Schwartz, Robert E; Mashimo, Hiroshi



Mesenchymal stem cells obtained after bone marrow transplantation or peripheral blood stem cell transplantation originate from host tissue.  


Mesenchymal stem cells (MSC) obtained from human bone marrow have been described as adult stem cells with the ability of extensive self-renewal and clonal expansion, as well as the capacity to differentiate into various tissue types and to modulate the immune system. Some data indicate that leukapheresis products may also contain non-hematopoietic stem cells, as they occur in whole bone marrow transplantation (BMT). However, there is still controversy whether MSC expand in the host after transplantation like blood progenitor cells do. Therefore, we were interested in finding out if graft MSC can be detected in leukapheresis products and in bone marrow after BMT and peripheral blood stem cell transplantation (PBSCT). Every sample from total bone marrow transplants exhibited growth of MSC after in vitro culture, but not one of nine leukapheresis products did. In addition, bone marrow aspirates of 9 patients receiving BMT and of 18 patients after PBSCT were examined for origin of MSC. Almost all MSC samples exhibited a complete host profile, whereas peripheral blood cells were of donor origin. We conclude that even if trace amounts of MSC are co-transplanted during PBSCT or BMT, they do not expand significantly in the host bone marrow. PMID:16132912

Dickhut, Andreas; Schwerdtfeger, Rainer; Kuklick, Larissa; Ritter, Markus; Thiede, Christian; Neubauer, Andreas; Brendel, Cornelia



A Rare Location for a Common Bone Tumor, Meta-Diaphyseal Giant Cell Tumor of Bone in an Adult Patient  

PubMed Central

Primary bone tumors can be either benign or malignant considering their natural history and cellular morphology. Benign bone tumors are much more frequent than malignant ones although some of them like giant cell tumor of bone can behave just like a malignant one that means has the capacity for massive local destruction and remote metastasis. Giant cell tumor of bone in adult people has a very strong and diagnostic predilection for epiphysial location in long bones. Very few cases have been so far reported for a giant cell tumor of bone with non-epiphysial location in a long bone.

Darioush, M. Barzi; H. Sami, Sam; Fallah, Ehsan



Simple methods for generating neural, bone and endodermal cell types from chick embryonic stem cells.  


Most work on embryonic stem cell differentiation uses mammalian cells derived from the blastocyst stage and some of the most widely used protocols to induce differentiation involve growing these cells in monolayer culture. Equivalent stem cells can be obtained from embryos of non-mammalian vertebrates, but to date this has only been successful in birds. These cells can contribute to all somatic lineages in chimaeras and can be induced to differentiate into a variety of cell types in vitro via embryoid body formation. However to date there are no reliable methods for differentiating them into descendants from each of the germ layers in monolayer culture, comparable to the protocols used in mammals. Here we describe three simple and reproducible protocols for differentiation of chick embryonic stem cells into mesoderm (bone), endoderm and neuroectoderm (neurons and glia) in monolayer culture. These methods open the way for more direct comparisons of the properties of mammalian and avian embryonic stem cells that may highlight similarities and differences. PMID:23047046

Boast, Sharon; Stern, Claudio D



A selective culture system for generating terminal deoxynucleotidyl transferase-positive (TdT+) lymphoid precursor cells in vitro. I. Description of the culture system  

PubMed Central

A primary xenogeneic culture system has been devised that selectively generates undifferentiated TdT+ lymphoblasts from rat bone marrow under conditions that do not support the growth or maintenance of rat colony- forming unit-spleen (CFU-S) or granulocyte/macrophage colony-forming cells (GM-CFC). The culture system requires a mouse bone marrow feeder layer, and a serum supplement that has markedly reduced levels of cortisol. The growth of TdT+ cells can be significantly enhanced by the addition of mesodermalizing factors (e.g., fibroblast growth factor, guinea pig bone marrow extract) to the culture medium, and the serum supplement can be decreased by the addition of selenium, transferrin, and T3. The cultured TdT+ cells are antigenically "null" cells that further resemble their normal counterparts in bone marrow with respect to morphology, size, cortisone sensitivity, and pattern of TdT fluorescence. The TdT+ cells are generated with equal facility from bone marrow of normal and congenitally athymic rats, can be maintained in logarithmic growth for at least 10 mos by serial passage in vitro, and do not cause leukemia when infused into irradiated recipients. Although the lineage relationships of these immature lymphoid cells have not yet been established, our working hypothesis, based on preliminary evidence, is that the cultured TdT+ cells are primitive members of the T cell series.



Reduced ischemic brain injury by partial rejuvenation of bone marrow cells in aged rats  

PubMed Central

Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases.

Taguchi, Akihiko; Zhu, Pengxiang; Cao, Fang; Kikuchi-Taura, Akie; Kasahara, Yukiko; Stern, David M; Soma, Toshihiro; Matsuyama, Tomohiro; Hata, Ryuji



Immunophenotypic characterization of human bone marrow endosteal cells.  


In order to determine the relationship between bone marrow (bm) endosteal cells (EDC) and hemopoietic progenitors, we have analyzed the immunophenotype of EDC using various antibodies (Ab) against mesenchymal antigens. The Ab were applied on paraffin sections of normal bm (iliac crest, n=17; talus, n=1; phalanx, n=1), myeloregenerative bm (after chemotherapy), and hematologic disorders (acute myeloid leukemia (AML), n=8; chronic myeloid leukemia (CML), n=6; myelodysplastic syndromes (MDS), n=14; severe aplastic anemia (SAA), n=4; essential thrombocythemia (ET), n=2; idiopathic (primary) osteomyelo-fibrosis (IMF), n=1; polycythemia vera (PV), n=1). In normal bm, EDC were found to react with Ab against vimentin, tenascin, alpha-smooth muscle actin, osteocalcin, CD51, and CD56, but did not react with Ab against CD3, CD15, CD20, CD34, CD45, CD68, or CD117. An identical phenotype of EDC was found in AML, MDS, SAA, ET, IMF, PV, myeloregenerative bm, and peripheral bones lacking active hemopoiesis (talus, phalanx). In patients with CML, EDC reacted with Ab to CD51, but did not react with Ab to CD56. Based on their unique antigen profile, EDC were enriched from normal bm by enzyme digestion and cell sorting. However, these enriched cells (CD56+, CD45-, CD34-) did not give rise to hemopoietic cells under the culture conditions used, i.e. in the presence of the growth factors IGF-1, bFGF, SCF, IL-3, and GM-CSF Together, our data do not support the hypothesis that EDC are totipotent mesenchymal progenitors giving rise to hemopoietic cells. PMID:10395106

Sillaber, C; Walchshofer, S; Mosberger, I; Gaiger, A; Simonitsch, I; Chott, A; Lechner, K; Valent, P



Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture.  


Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine. PMID:23798652

Laydi, F; Rahouadj, R; Cauchois, G; Stoltz, J-F; de Isla, N



Engipore acts on human bone marrow stem cells  

PubMed Central

Objectives Porous HA scaffolds are promising materials for tissue engineering because they offer a tridimensional support and serve as template for cell proliferation and at last tissue formation. Engipore provide a natural 3D scaffold with organic fibrous material in bone. However, how this material alters osteoblast activity to promote bone formation is poorly understood. Materials and methods To study how Engipore can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed. Results Engipore causes a significant induction of osteoblast transcriptional factors like SP7 and RUNX2 and of the bone-related gene osteocalcin (BGLAP). The expression of CD105 was not significantly changed in stem cells treated with Engipore with respect to untreated cells, while SSP1 (osteopontin) was significantly down expressed thus reducing osteoclast activity. Conclusions The obtained results can be relevant to better understand the molecular mechanism of bone regeneration.

Sollazzo, Vincenzo; Palmieri, Annalisa; Girardi, Ambra; Farinella, Francesca; Carinci, Francesco



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

Hombauer, H; Minguell, J J



Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds.  


The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair. PMID:11890685

Partridge, Kris; Yang, Xuebin; Clarke, Nicholas M P; Okubo, Yasunori; Bessho, Kazuhisa; Sebald, Walter; Howdle, Steven M; Shakesheff, Kevin M; Oreffo, Richard O C



Oscillatory behavior of cells in tissue culture  

Microsoft Academic Search

Fibroblasts and epithelial cells organize themselves in distinct patterns in tissue culture which indicates that neighboring cells communicate. A striking example of such communication is the oscillatory behavior of Madin-Darby canine kidney (MDCK) cells reported here. These oscillations were discovered using a biosensor referred to as ECIS (Electric Cell-substrate Impedance Sensing). In this measurement cells are seeded out on a

Ivar Giaever; Michael F. A. Linton; Charles R. Keese



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



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

PubMed Central

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

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



Rat bone marrow stromal cells could be induced into Schwann cell precursor-like cells in vitro.  


Schwann cells (SC) which are myelin forming cells in peripheral nervous system can remyelinate demyelinated central nervous system (CNS) axons in vivo. However, potential drawbacks to the use of SC in the CNS are nevertheless apparent, and Schwann cell precursors (SCP) are favourable cells for myelin repair in the CNS. But for clinical use, it is difficult to obtain sufficient large number of SCP. In the present study, rat bone marrow stromal cells (MSCs) were cultured, identified and then converted into neurospheres. Then neurospheres were identified and induced into SCP-like cells. SCP-like cells were flattened in shape, p75(+)GFAP(-)S-100(-)nestin(-), and could differentiate into SC-like cells, similar to genuine SCP. Our data suggested that MSCs could be induced into SCP-like cells. PMID:21056626

Xu, Yongfeng; Xiong, Fu; Liu, Lan; Zhang, Cheng



Cocoa bean cell and embryo culture  

Microsoft Academic Search

Callus culture of cocoa bean was initiated from immature cotyledons on agar medium. By dispersing these callus cells, a liquid\\u000a suspension culture was established. The lipid composition of cocoa suspension culture was investigated and compared with those\\u000a of cocoa beans of different maturities. Factors affecting fatty acid and triglyceride synthesis in cocoa suspension cultures\\u000a also were studied. In parallel studies,

Ming-Che Wen; Bruce German; John E. Kinsella



Bone marrow cells from allogeneic bone marrow chimeras inhibit the generation of cytotoxic lymphocyte responses against both donor and recipient cells  

SciTech Connect

When added to a mixed lymphocyte culture, bone marrow cells suppress the generation of CTL activity against H-2 Ag shared by the BM cells and the stimulator cells. These cells have been referred to as veto cells and are thought to play a role in maintaining self-tolerance. We analyzed the H-2 specificity of the suppression expressed by the veto cells from H-2 incompatible bone marrow chimeras, because lymphocytes of such chimeras had been shown to be tolerant to both donor and recipient Ag when tested by CTL responses. We found that the bone marrow cells of such chimeras which were featured by non-T and non-B cell characteristics inhibited the generation of CTL directed against either donor or recipient Ag, but not against third-party Ag. These observations suggest that in allogeneic chimeras the veto or veto-like cells alter the inhibitory specificity exhibited in the recipient microenvironment and indicate that these cells are directly involved in the induction and maintenance of self-tolerance.

Ogasawara, M.; Iwabuchi, K.; Good, R.A.; Onoe, K.




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.

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.



Development of silk-based scaffolds for tissue engineering of bone from human adipose derived stem cells  

PubMed Central

Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and demonstrated ability to support bone formation, in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASC), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol - HFIP), pore sizes (250–500?m vs. 500–1000?m) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes with decellularized trabecular bone (DCB) included as a “gold standard”. The scaffolds were seeded with hASC and cultured for 7 weeks in osteogenic media. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600?m porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2, and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.

Correia, Cristina; Bhumiratana, Sarindr; Yan, Le-Ping; Oliveira, Ana L.; Gimble, Jeffrey M.; Rockwood, Danielle; Kaplan, David L.; Sousa, Rui A.; Reis, Rui L.; Vunjak-Novakovic, Gordana



Isolation of rabbit bone marrow mesenchymal stem cells using density gradient centrifugation and adherence screening methods.  


Aim: Under special conditions, bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and chondroblasts. However, MSCs are few in bone marrow. How to harvest, purify and rapidly proliferate in vitro is a foundation of application in tissue engineering technique. To optimize, collect, purify, assess rabbit BMSCs and to observe the biological character of BMSCs. Methods: Two female New Zealand rabbits aged 2 months were used for MSC collection and primary culture. Bone marrow solution was purified by density gradient centrifugation and adherence screening method. Culture solution was obtained. BMSCs were incubated in phosphate buffered solution (PBS), supplemented with 2.5 g/L trypsin (3.0 mL), and placed in an incubator at 37 °C for two or three minutes. Cell morphology was observed using an inverted microscope. The digestion was stopped when cytoplasm recovery, long and thin cells with large intercellular space, and few round cells appeared. Subsequently, BMSCs were incubated in serum-free L-DMEM, and placed in a plastic culture flask at 1.0×108/L. MSC morphology, ultrastructure and surface marker; proliferation of the first, third, fifth, eighth and tenth passages of BMSCs; cell growth curve was drawn. BMSCs was pure following density gradient centrifugation and adherence screening method. The third and fifth passage of cells had typical whirlpool-shape. Results and conclusion: Transmission electron microscope demonstrated that round or oval MSCs possessed large nuclei, big nucleus proportion, a few cellular organ. These were low-differentiated cells. Growth curve of cultured MSCs was "S" shape. The first, third and fifth passage cells had strong reproductive capability. The eighth and tenth passage of cells had significantly reduced proliferation. Cells isolated were positive for CD44 and CD90, but negative for CD34. These were low-differentiated cells under the electron microscope. Isolated cells are MSCs, with the property of stem cells. The third and fifth passage cells are pure, with strong reproductive capability. PMID:24101109

Xia, C S; Zuo, A J; Wang, C Y; Wang, Y Z



Lethal graft-versus-host disease: modification with allogeneic cultured donor cells  

SciTech Connect

The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta-positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90-day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences.

Mauch, P.; Lipton, J.M.; Hamilton, B.; Obbagy, J.; Kudisch, M.; Nathan, D.; Hellman, S.



Renal repair: role of bone marrow stem cells  

Microsoft Academic Search

Acute kidney injury carries severe consequences and has limited treatment options. Bone marrow stem cells may offer the potential\\u000a for treatment of acute kidney injury. The purpose of this review is twofold. The first purpose is to provide a concise overview\\u000a of the biology of bone marrow stem cells, including hematopoietic stem cells and mesenchymal stem cells, for clinical nephrologists

Fangming Lin



Stimulation of bone resorption and cell proliferation in vitro by human gingival fibroblasts from patients with periodontal disease.  


In the present communication we report that fibroblasts, isolated from human gingiva obtained from 13 different patients, secreted soluble product(s) which can promote bone resorption in vitro. Fibroblasts were isolated from explants of human gingiva, subcultured, grown to confluent monolayers, subsequently cultured in growth arrest media for 0-72 h and conditioned media harvested. Bone resorption was assessed in cultured mouse calvarial bone by quantifying the mobilization of minerals and the release of lysosomal enzymes. Human fibroblast-conditioned media (HFCM) dose-dependently stimulated the release of 45Ca from prelabelled bones and the mobilization of stable calcium and inorganic phosphate from unlabelled bones. In addition, HFCM increased the release of beta-glucuronidase and beta-N-acetylglucosaminidase from the calvaria. No effect of HFCM on the release of 45Ca from dead bones could be seen. HFCM caused a dose-dependent increased degradation of bone matrix proteins, as assessed by the release of 3H from [3H]proline-labelled calvaria. The stimulation of 45Ca release could already be seen after 3-12 h of treatment. Treatment of the bones with HFCM for 12 h was sufficient to obtain a prolonged stimulation of 45Ca release. Bones cultured in the presence of HFCM showed an increased number of osteoclasts. Calcitonin, but not indomethacin, inhibited 45Ca release stimulated by HFCM. Ultrafiltration of HFCM did not cause any loss of the 45Ca release response. The amount of bone-resorbing activity produced by the gingival cells was proportional to the number of cells. In addition, HFCM stimulated the proliferation of human fibroblasts and osteoblast-enriched mouse calvarial bone cells. It is concluded that human gingival fibroblasts secrete one or several factors that can stimulate osteoclastic bone resorption in vitro by a prostaglandin-independent pathway. PMID:2224207

Lerner, U H; Hänström, L; Sjöström, S



Chloroquine, hydroxystilbamidine, and dapsone inhibit resorption of fetal rat bone in organ culture  

Microsoft Academic Search

Summary  Three potential inhibitors of lysosomal enzyme release, chloroquine, hydroxystilbamidine, and dapsone were tested for their\\u000a effects on the release of previously incorporated45Ca and beta (?)-glucuronidase from fetal rat long bones cultured in a chemically defined medium. At concentrations of 10?5 to 10?8M, all three agents were able to inhibit the stimulation of bone resorption by parathyroid hormone (PTH) or prostaglandin

Gabriel Eilon; Lawrence G. Raisz



Lactate Transport in Cultured Glial Cells  

Microsoft Academic Search

Uptake of L-lactate was investigated with a radioactive tracer method in cultured rat glioma cells and in astroglia-rich primary cultures derived from rat brain. In the glioma cells, a saturable component of uptake was identified with half-maximal uptake occurring at 1.0±0.4 mM lactate. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. In astroglia-rich primary cultures,

Ralf Dringen; Hajo Peters; Heinrich Wiesinger; Bernd Hamprecht



Glucosylation of taxifolin with cultured plant cells.  


Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus. PMID:23980419

Shimoda, Kei; Kubota, Naoji; Hamada, Manabu; Sugamoto, Masahiro; Ishihara, Kohji; Hamada, Hatsuyuki; Hamada, Hiroki



Crustacean primary cell culture: A technical approach  

Microsoft Academic Search

Crustacean cell culture has gained attention as a potent model to assist in the development of diagnostic reagents and probes for use in the shrimp, crayfish and lobster industries. The availability of such cellular tools is especially important to industries which use intensive aquaculture methods and thus have increased risk of disease problems. Indeed, crustacean cell cultures offer potential for

Jean-Yves Toullec



In vitro studies with melphalan and pediatric neoplastic and normal bone marrow cells.  


Chemotherapeutic agents are used in increasingly high dosages to treat patients with refractory cancers. An in vitro clonogenic assay was used to investigate the effects of melphalan on cultures of human neuroblastoma, Ewing's sarcoma, and osteosarcoma cells, as well as on normal bone marrow cells. A 1-hr incubation with 10(-5) M melphalan significantly inhibited tumor colony growth of both fresh neuroblastoma and osteosarcoma cells (p less than 0.01) while Ewing's sarcoma cells and normal bone marrow appeared resistant to melphalan even at a 100-fold higher concentration. Incubation with melphalan for 8 hr did not significantly increase the sensitivity of neuroblastoma and osteosarcoma or the relative resistance of Ewing's sarcoma cells; however, normal bone marrow which had remained resistant to melphalan after 1 hr of incubation, showed significant inhibition of colony growth after an 8-hr incubation with the agent. Repeated exposure to melphalan increased the degree of inhibition of both tumor and normal marrow colonies. Fresh neuroblastoma cells were significantly more sensitive than long-term cultured neuroblastoma cells at all drug doses tested. HPLC studies demonstrated that the loss of melphalan followed first-order kinetics with a half-life of 69 min, and that in addition to the 2 known breakdown products, mono- and dihydroxy-melphalan, several other peaks were present which were not attributable to the tissue culture medium or the buffer solutions. PMID:3458679

Worthington-White, D A; Graham-Pole, J R; Stout, S A; Riley, C M



Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells  

PubMed Central

To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM) was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering.

Blois, Anna L; Xue, Ying; Xing, Zhe; Cottler-Fox, Michele; Fristad, Inge; Leknes, Knut N; Lorens, James B; Mustafa, Kamal



Effects of a bone resorptive factor from human cancer ascites fluid on rat bone cell calcium and cyclic AMP  

Microsoft Academic Search

Summary  The effects of a bone resorptive protein isolated from human cancer ascites fluid on bone cell calcium and cyclic AMP were\\u000a studied with fetal rat cells. The osteoclast-activating factor increased bone cell calcium uptake at 37°C and 4°C with no\\u000a direct effects on calcium efflux. Concentrations of the resorptive factor that increased in vitro bone resorption and cell\\u000a calcium uptake

R. Dziak; Y. W. Chang; D. Humphries; W. Lloyd; H. Wells; K. Schmid; R. B. Nimberg



Bone marrow cells of swine: collection and separation.  


Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy. PMID:22362561

Branco, Érika; Cabral, Rosa; Gomes, Bruno Duarte; Kfoury, José Roberto; Miglino, Maria Angelica



21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...2009-04-01 false Cultured animal and human cells. 864.2280 Section 864...864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell...



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...864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell...