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Sample records for bone cell cultures

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

    PubMed

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

    1997-09-01

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

  2. The Effect of Spaceflight on Bone Cell Cultures

    NASA Technical Reports Server (NTRS)

    Landis, William J.

    1999-01-01

    Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural

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

    PubMed

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

    2008-12-01

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

  4. Comparisons of mouse mesenchymal stem cells in primary adherent culture of compact bone fragments and whole bone marrow.

    PubMed

    Cai, Yiting; Liu, Tianshu; Fang, Fang; Xiong, Chengliang; Shen, Shiliang

    2015-01-01

    The purification of mouse bone marrow mesenchymal stem cells (BMSCs) by using the standard method of whole bone marrow adherence to plastic still remains ineffective. An increasing number of studies have indicated compact bone as an alternative source of BMSCs. We isolated BMSCs from cultured compact bone fragments and investigated the proliferative capacity, surface immunophenotypes, and osteogenic and adipogenic differentiations of the cells after the first trypsinization. The fragment culture was based on the fact that BMSCs were assembled in compact bones. Thus, the procedure included flushing bone marrow out of bone cavity and culturing the fragments without any collagenase digestion. The cell yield from cultured fragments was slightly less than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90 and CD44 expressions and less CD45 expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than those of cells from bone marrow. The directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture.

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

    PubMed

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

    1998-12-01

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

  6. Spaceflight effects on cultured embryonic chick bone cells

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

    A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the

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

    SciTech Connect

    Miyahara, Tatsuro; Takata, Masakazu; Miyata, Masaki; Nagai, Miyuki; Sugure, Akemi; Kozuka, Hiroshi; Kuze, Shougo )

    1991-08-01

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

  8. The Impairment of Osteogenesis in Bone Sialoprotein (BSP) Knockout Calvaria Cell Cultures Is Cell Density Dependent

    PubMed Central

    Bouet, Guenaelle; Bouleftour, Wafa; Juignet, Laura; Linossier, Marie-Thérèse; Thomas, Mireille; Vanden-Bossche, Arnaud; Aubin, Jane E.; Vico, Laurence; Marchat, David; Malaval, Luc

    2015-01-01

    Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors. PMID:25710686

  9. The preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation.

    PubMed

    Kroeger, Kelly; Collins, Michelle; Ugozzoli, Luis

    2009-01-01

    It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed. PMID:19229174

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells.

    PubMed

    Gurel Pekozer, Gorke; Torun Kose, Gamze; Hasirci, Vasif

    2016-11-01

    Co-culture of bone forming cells and endothelial cells to induce pre-vascularization is one of the strategies used to solve the insufficient vascularization problem in bone tissue engineering attempts. In the study, primary cells isolated from 2 different tissues of the same animal, rat bone marrow stem cells (RBMSCs) and rat aortic endothelial cells (RAECs) were co-cultured to study the effects of co-culturing on both osteogenesis and angiogenesis. The formation of tube like structure in 2D culture was observed for the first time in the literature by the co-culture of primary cells from the same animal and also osteogenesis and angiogenesis were investigated at the same time by using this co-culture system. Co-cultured cells mineralized and formed microvasculature beginning from 14days of incubation. After 28days of incubation in the osteogenic medium, expression of osteogenic genes in co-cultures was significantly upregulated compared to RBMSCs cultured alone. These results suggest that the co-culture of endothelial cells with mesenchymal stem cells induces both osteogenesis and angiogenesis.

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

    SciTech Connect

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

    1985-05-01

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

  15. Bone culture research

    NASA Technical Reports Server (NTRS)

    Partridge, Nicola C.

    1993-01-01

    The experiments described are aimed at exploring PTH regulation of production of collagenase and protein inhibitors of collagenase (tissue inhibitors of metalloproteases, TIMP-1 and -2) by osteoblast-like osteosarcoma cells under conditions of weightlessness. The results of this work will contribute to information as to whether a microgravity environment alters the functions and responsiveness of the osteoblast. The objectives of the Bone Culture Research (BCR) experiment are: to observe the effects of microgravity on the morphology, rate of proliferation, and behavior of the osteoblastic cells, UMR 106-01; to determine whether microgravy affects the hormonal sensitivity of osteroblastic cells; and to measure the secretion of collagenase and its inhibitors into the medium under conditions of microgravity. The methods employed will consist of the following: the osteoblast-like cells, UMR-106-01, will be cultured in four NASDA cell culture chambers; two chambers will be subjected to microgravity on SL-J; two chambers will remain on the ground at KSC as ground controls but subjected to an identical set of culture conditions as on the shuttle; media will be changed four times; twice the cells will receive the hormone parathyroid hormone-related protein (PTHrP) and media collected; cells will be photographed under conditions of microgravity; and media and photographs will be analyzed upon return to determine whether functions of the cells changed.

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

    SciTech Connect

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

    1990-08-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    1994-02-01

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

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

    PubMed

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

    2005-01-01

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

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

    PubMed

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

    2014-12-16

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  3. In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

    PubMed Central

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

    2014-01-01

    Abstract Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano

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

    PubMed

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

    2014-12-01

    Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography

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

    PubMed

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

    2014-12-01

    Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography

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

    SciTech Connect

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

    2005-09-30

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

  7. Cytocompatibility of the selected calcium phosphate based bone cements: comparative study in human cell culture.

    PubMed

    Olkowski, Radosław; Kaszczewski, Piotr; Czechowska, Joanna; Siek, Dominika; Pijocha, Dawid; Zima, Aneta; Ślósarczyk, Anna; Lewandowska-Szumieł, Małgorzata

    2015-12-01

    Calcium phosphate cements (CPC) are valuable bone fillers. Recently they have been also considered as the basis for drug-, growth factors- or cells-delivery systems. Broad possibilities to manipulate CPC composition provide a unique opportunity to obtain materials with a wide range of physicochemical properties. In this study we show that CPC composition significantly influences cell response. Human bone derived cells were exposed to the several well-characterized different cements based on calcium phosphates, magnesium phosphates and calcium sulfate hemihydrate (CSH). Cell viability assays, live/dead staining and real-time observation of cells in contact with the materials (time-laps) were performed. Although all the investigated materials have successfully passed a standard cytocompatibility assay, cell behavior in a direct contact with the materials varied depending on the material and the experimental system. The most recommended were the α-TCP-based materials which proved suitable as a support for cells in a direct contact. The materials which caused a decrease of calcium ions concentration in culture induced the negative cell response, however this effect might be expected efficiently compensated in vivo. All the materials consisting of CSH had negative impact on the cells. The obtained results strongly support running series of cytocompatibility studies for preclinical evaluation of bone cements. PMID:26511138

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

    SciTech Connect

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

    1986-01-01

    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. Furthermore, it has been suggested that calciotropic hormones may express their actions on bone cells by modulating the messenger functions of intracellular Ca. Before these postulates can be more fully tested, the homeostasis of intracellular Pb and Ca must be understood more clearly. We have designed experiments to characterize the steady-state kinetic distribution and behavior of /sup 210/Pb and /sup 45/Ca in osteoclastic bone cells and to identify biological structures or functions associated with the kinetic pools. Cultures were labeled with /sup 210/Pb or /sup 45/Ca and the kinetic parameters were obtained by analysis of isotope washout curves. Cellular metabolism was defined by three kinetic pools of intracellular Pb and Ca. The largest and slowest exchanging pool was characterized as a mitochondrial compartment for both metals. These data indicate that Pb and Ca share similar intracellular pathways in osteoclastic bone cells and that both metals are readily exchangeable in this in vitro system. The results of other experiments have revealed that lead, at relatively low medium concentrations, produces a marked increase in all three intracellular Ca compartments. 18 refs., 2 figs.

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

    PubMed

    Pacak, Christina A; Cowan, Douglas B

    2014-01-01

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

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

    PubMed

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

    2007-01-01

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

  11. Extended Culture Conditions for Multipotent Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Zhang, Kui; Ikeda, Yayoi; Kasugai, Shohei; Ikeda, Masa-Aki

    2016-03-01

    Mesenchymal stem cells (MSCs) offer a promising source of cells for musculoskeletal regeneration because of their potential to differentiate into bone, cartilage and fat. However, their proliferation and multilineage differentiation potential decreases with aging or increased time in in vitro culture. To determine culture conditions capable of enabling maintenance of MSCs for extended periods of time, human bone marrow-derived MSCs (BM-MSCs) were cultured in growth medium containing various combinations of growth factors and small chemical compounds. Upon reaching confluence, MSCs were subcultured continuously and then tested for differentiation capacity. After screening various growth factors and small chemical compounds, we found a combination capable of maintaining the proliferation potential of BM-MSCs obtained from a 19-year-old donor (young MSCs) up to passage 13 (P13). In contrast, unsupplemented MSCs reached senescence at P10. Total population doublings of control (P10) and supplemented MSCs (P12) were estimated at 20.4 and 42, respectively. Young MSCs cultured with supplements maintained osteogenic, adipogenic and chondrogenic differentiation capacities at P12 as confirmed by expression of lineage-specific differentiation markers. Furthermore, the supplementation of to BM-MSCs obtained from 65- and 79-year-old donors (aged MSCs) also continued to proliferate until P12, and maintained osteogenic and adipogenic differentiation capacity until P7 and P8, respectively, whereas, unsupplemented aged MSCs stopped proliferating at P8. These results indicate that our extended culture conditions maintained the proliferative capacity of young MSCs while retaining their multipotent differentiation potential, and improved both proliferation and differentiation of aged MSCs.

  12. Extended Culture Conditions for Multipotent Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Zhang, Kui; Ikeda, Yayoi; Kasugai, Shohei; Ikeda, Masa-Aki

    2016-03-01

    Mesenchymal stem cells (MSCs) offer a promising source of cells for musculoskeletal regeneration because of their potential to differentiate into bone, cartilage and fat. However, their proliferation and multilineage differentiation potential decreases with aging or increased time in in vitro culture. To determine culture conditions capable of enabling maintenance of MSCs for extended periods of time, human bone marrow-derived MSCs (BM-MSCs) were cultured in growth medium containing various combinations of growth factors and small chemical compounds. Upon reaching confluence, MSCs were subcultured continuously and then tested for differentiation capacity. After screening various growth factors and small chemical compounds, we found a combination capable of maintaining the proliferation potential of BM-MSCs obtained from a 19-year-old donor (young MSCs) up to passage 13 (P13). In contrast, unsupplemented MSCs reached senescence at P10. Total population doublings of control (P10) and supplemented MSCs (P12) were estimated at 20.4 and 42, respectively. Young MSCs cultured with supplements maintained osteogenic, adipogenic and chondrogenic differentiation capacities at P12 as confirmed by expression of lineage-specific differentiation markers. Furthermore, the supplementation of to BM-MSCs obtained from 65- and 79-year-old donors (aged MSCs) also continued to proliferate until P12, and maintained osteogenic and adipogenic differentiation capacity until P7 and P8, respectively, whereas, unsupplemented aged MSCs stopped proliferating at P8. These results indicate that our extended culture conditions maintained the proliferative capacity of young MSCs while retaining their multipotent differentiation potential, and improved both proliferation and differentiation of aged MSCs. PMID:27443069

  13. Ultrastructural study of cultured ovine bone marrow-derived mesenchymal stromal cells.

    PubMed

    Desantis, Salvatore; Accogli, Gianluca; Zizza, Sara; Mastrodonato, Maria; Blasi, Antonella; Francioso, Edda; Rossi, Roberta; Crovace, Antonio; Resta, Leonardo

    2015-09-01

    Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify "in situ" the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected.

  14. Ultrastructural study of cultured ovine bone marrow-derived mesenchymal stromal cells.

    PubMed

    Desantis, Salvatore; Accogli, Gianluca; Zizza, Sara; Mastrodonato, Maria; Blasi, Antonella; Francioso, Edda; Rossi, Roberta; Crovace, Antonio; Resta, Leonardo

    2015-09-01

    Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify "in situ" the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected. PMID:26196242

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

    PubMed

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

    2013-12-01

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

  16. Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

    PubMed

    Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

    2015-04-01

    The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture.

  17. Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

    PubMed Central

    Beertsen, W; Heersche, JNM; Aubin, JE

    1982-01-01

    Free and polymerized tubulin were measured in bone cells and Chinese hamster ovary (CHO) cells cultured on plastic substrata. Polymerized tubulin was stabilized in a microtubule- stabilizing medium (MSM) containing 50 percent glycerol and separated from free tubulin by centrifugation. Tubulin content was assayed in both fractions by the colchicines- binding assay. The measured degree of polymerization in both bone cells and CHO cells varied with stabilixation conditions. The degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in attached cells was found to increase up to 73 percent during the first 20 min after addition of the MSM at 24 degrees C, and remained constant thereafter. Stabilization of 0 degrees C resulted in a decrease down to 62 percent in the degree of constant thereafter. Stabilization at 0 degrees C resulted in a decrease down to 62 percent in the degree of polymerization during the first 20 min after addition of the MSM at 24 degrees C, and remained constant thereafter. Confluent bone cells maintained at 0 degrees C for 1 h before stabilization contained significantly less polymerized tubulin than control cells kept at 37 degrees C using stabilization both at 0 degrees C and at 24 degrees C. Changes in bone cell morphology induced by incubation of cells with prostaglandin E(1) or E(2), parthyroid hormone, and dibutyryl cyclic AMP were not associated with a change in the degree of tubulin polymerization. This was confirmed morphologically by immunofluorescence using affinity-purified tubulin antibodies: microtubules in hormone- treated cells were not noticeably reorganized when compared to microtubule organization in control cells. They were, however, squeezed closer together in cellular pseudopods due to the altered cell shape. This altered cell shape appears to be correlated

  18. Human periodontal ligament stem cells cultured onto cortico-cancellous scaffold drive bone regenerative process.

    PubMed

    Diomede, F; Zini, N; Gatta, V; Fulle, S; Merciaro, I; D'Aurora, M; La Rovere, R M; Traini, T; Pizzicannella, J; Ballerini, P; Caputi, S; Piattelli, A; Trubiani, O

    2016-01-01

    The purpose of this work was to test, in vitro and in vivo, a new tissue-engineered construct constituted by porcine cortico-cancellous scaffold (Osteobiol Dual Block) (DB) and xeno-free ex vivo culture of human Periodontal Ligament Stem Cells (hPDLSCs). hPDLSCs cultured in xeno-free media formulation preserved the stem cells' morphological features, the expression of stemness and pluripotency markers, and their ability to differentiate into mesenchymal lineage. Transmission electron microscopy analysis suggested that after one week of culture, both noninduced and osteogenic differentiation induced cells joined and grew on DB secreting extracellular matrix (ECM) that in osteogenic induced samples was hierarchically assembled in fibrils. Quantitative RT-PCR (qRT-PCR) showed the upregulation of key genes involved in the bone differentiation pathway in both differentiated and undifferentiated hPDLSCs cultured with DB (hPDLSCs/DB). Functional studies revealed a significant increased response of calcium transients in the presence of DB, both in undifferentiated and differentiated cells stimulated with calcitonin and parathormone, suggesting that the biomaterial could drive the osteogenic differentiation process of hPDLSCs. These data were confirmed by the increase of gene expression of L-type voltage-dependent Ca2+ (VDCCL), subunits α1C and α2D1 in undifferentiated cells in the presence of DB. In vivo implantation of the hPDLSCs/DB living construct in the mouse calvaria evidenced a precocious osteointegration and vascularisation process. Our results suggest consideration of DB as a biocompatible, osteoinductive and osteoconductive biomaterial, making it a promising tool to regulate cell activities in biological environments and for a potential use in the development of new custom-made tissue engineering. PMID:27633707

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

    PubMed

    Muscari, Claudio; Gamberini, Chiara; Basile, Ilaria; Bonafé, Francesca; Valgimigli, Simond; Capitani, Ombretta; Guarnieri, Carlo; Caldarera, Claudio Marcello

    2010-02-06

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

  20. A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering.

    PubMed

    Zhang, Zhi-Yong; Teoh, Swee Hin; Teo, Erin Yiling; Khoon Chong, Mark Seow; Shin, Chong Woon; Tien, Foo Toon; Choolani, Mahesh A; Chan, Jerry K Y

    2010-11-01

    Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-06-01

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

  5. DT56a stimulates gender-specific human cultured bone cells in vitro.

    PubMed

    Somjen, Dalia; Katzburg, Sara; Lieberherr, M; Hendel, David; Yoles, Israel

    2006-01-01

    DT56a found to have SERM-like properties is used for the treatment of menopausal symptoms and osteoporosis. In vivo experiments demonstrated that DT56a displayed selective estrogenic activity; it stimulated creatine kinase (CK) specific activity in the skeletal tissues but not on the uterus of ovariectomized rats. DT56a, when applied together with estradiol-17beta (E(2)), completely inhibited the E(2)-stimulated CK, as demonstrated by other SERMs. DT56a stimulated bone formation in a rat model as measured by histological and histomorphometrical parameters. In a clinical study, administration of DT56a (Femarelle) resulted in a considerable elevation of bone mineral density and relief of menopausal symptoms. The aim of the present study was to analyze the effects of DT56a in vitro on human-derived bone cultured osteoblasts (Ob), by measuring its effects, at different concentrations, on DNA synthesis, CK and alkaline phosphatase (ALP) specific activities as well as changes in intracellular [Ca(2+)](i) concentrations. DT56a stimulated CK and DNA synthesis in both pre- and post-menopausal female Ob with maximal effect at 100 ng/ml for both age groups. In addition, DT56a stimulated ALP in Ob from both pre- and post-menopausal women with maximal effect at lower dose of 50 ng/ml, with higher response of pre-menopausal cells. Raloxifene (Ral) inhibited all DT56a-stimulated changes in Ob from both age groups. DT56a, when given together with E(2), completely antagonized E(2)-stimulated effects demonstrating its nature as a phyto-SERM. DT56a also, dose dependency, stimulated the intracellular levels of [Ca(2+)](i) with maximal effect at 10 ng/ml. Male-derived Ob did not respond to DT56a in any parameter. In summary, DT56a stimulated sex-specifically female-derived Ob, indicating its unique nature compared to the compounds currently used for postmenopausal osteoporosis by being bone-forming and not only an anti-resorptive agent.

  6. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  7. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

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

    PubMed Central

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

    2009-01-01

    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 γ-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. PMID:19450750

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

    SciTech Connect

    Perkins, S.; Fleischman, R.A.

    1988-04-01

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

  10. 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.

    PubMed

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

    2010-07-01

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

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

    PubMed

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

    2016-05-01

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

  12. Cell therapy for bone repair.

    PubMed

    Rosset, P; Deschaseaux, F; Layrolle, P

    2014-02-01

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

  13. Histological and Immunohistochemical Evaluation of Autologous Cultured Bone Marrow Mesenchymal Stem Cells and Bone Marrow Mononucleated Cells in Collagenase-Induced Tendinitis of Equine Superficial Digital Flexor Tendon

    PubMed Central

    Crovace, Antonio; Lacitignola, Luca; Rossi, Giacomo; Francioso, Edda

    2010-01-01

    The aim of this study was to compare treatment with cultured bone marrow stromal cells (cBMSCs), bone marrow Mononucleated Cells (BMMNCs), and placebo to repair collagenase-induced tendinitis in horses. In six adult Standardbred horses, 4000 IU of collagenase were injected in the superficial digital flexor tendon (SDFT). Three weeks after collagenase treatment, an average of either 5.5 × 106 cBMSCs or 1.2 × 108 BMMNCs, fibrin glue, and saline solution was injected intralesionally in random order. In cBMSC- and BMMNCS-treated tendons, a high expression of cartilage oligomeric matrix protein (COMP) and type I collagen, but low levels of type III collagen were revealed by immunohistochemistry, with a normal longitudinally oriented fiber pattern. Placebo-treated tendons expressed very low quantities of COMP and type I collagen but large numbers of randomly oriented type III collagen fibers. Both cBMSC and BMMNCS grafts resulted in a qualitatively similar heling improvement of tendon extracellular matrix, in terms of the type I/III collagen ratio, fiber orientation, and COMP expression. PMID:20445779

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

    SciTech Connect

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

    2010-04-02

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

  15. Microgravity and bone cell mechanosensitivity.

    PubMed

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

    2003-01-01

    The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Bone not only develops as a structure designed specifically for mechanical tasks, but it can adapt during life toward more efficient mechanical performance. Mechanical adaptation of bone is a cellular process and needs a biological system that senses the mechanical loading. The loading information must then be communicated to the effector cells that form new bone or destroy old bone. The in vivo operating cell stress derived from bone loading is likely the flow of interstitial fluid along the surface of osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin (PG) synthesis and expression of prostaglandin G/H synthase inducible cyclooxygenase (COX-2). Cultured bone cells also rapidly produce nitric oxide (NO) in response to fluid flow as a result of activation of endothelial nitric oxide synthase (ecNOS), which enzyme also mediates the adaptive response of bone tissue to mechanical loading. Earlier studies have shown that the disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction. Microgravity, or better near weightlessness, is associated with the loss of bone in astronauts, and has catabolic effects on mineral metabolism in bone organ cultures. This might be explained as resulting from an exceptional form of disuse under near weightlessness conditions. However, under near weightlessness conditions the assembly of cytoskeletal elements may be altered since it has been shown that the direction of the gravity vector determines microtubular pattern formation in vivo. We found earlier that the transduction of mechanical signals in bone cells also involves the cytoskeleton and is related to PGE2 production. Therefore it is possible that the

  16. Microgravity and bone cell mechanosensitivity

    NASA Astrophysics Data System (ADS)

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

    2003-10-01

    The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Bone not only develops as a structure designed specifically for mechanical tasks, but it can adapt during life toward more efficient mechanical performance. Mechanical adaptation of bone is a cellular process and needs a biological system that senses the mechanical loading. The loading information must then be communicated to the effector cells that form new bone or destroy old bone. The in vivo operating cell stress derived from bone loading is likely the flow of interstitial fluid along the surface of osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin (PG) synthesis and expression of prostaglandin G/H synthase inducible cyclooxygenase (COX-2). Cultured bone cells also rapidly produce nitric oxide (NO) in response to fluid flow as a result of activation of endothelial nitric oxide synthase (ecNOS), which enzyme also mediates the adaptive response of bone tissue to mechanical loading. Earlier studies have shown that the disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction. Microgravity, or better near weightlessness, is associated with the loss of bone in astronauts, and has catabolic effects on mineral metabolism in bone organ cultures. This might be explained as resulting from an exceptional form of disuse under near weightlessness conditions. However, under near weightlessness conditions the assembly of cytoskeletal elements may be altered since it has been shown that the direction of the gravity vector determines microtubular pattern formation in vivo. We found earlier that the transduction of mechanical signals in bone cells also involves the cytoskeleton and is related to PGEZ production. Therefore it is possible that the

  17. Human alveolar bone-derived cell-culture behaviour on biodegradable poly(L-lactic Acid).

    PubMed

    Bombonato-Prado, Karina Fittipaldi; Wimmers Ferreira, Maidy Redher; Rosa, Adalberto Luiz; de Oliveira, Paulo Tambasco; Jahno, Vanusca Dalosto; da Silva, Jefferson Braga; Ligabue, Rosane; Einloft, Sandra

    2009-01-01

    Poly(L-lactic acid) (PLA) is a polymer of great technological interest, whose excellent mechanical properties, thermal plasticity and bioresorbability render it potentially useful for environmental applications, as a biodegradable plastic and as a biocompatible material in biomedicine. The interactions between an implant material surface and host cells play central roles in the integration, biological performance and clinical success of implanted biomedical devices. Osteoblasts from human alveolar bone were chosen to investigate the cell behaviour when in contact with PLA discs. Cell morphology and adhesion through osteopontin (OPN) and fibronectin (FN) expression were evaluated in the initial osteogenesis, as well as cell proliferation, alkaline phosphatase activity and bone nodule formation. It was shown that the polymer favoured cell attachment. Cell proliferation increased until 21 days but in a smaller rate when compared to the control group. On the other hand, ALP activity and bone mineralization were not enhanced by the polymer. It is suggested that this polymer favours cell adhesion in the early osteogenesis in vitro, but it does not enhance differentiation and mineralization.

  18. Microgravity and Bone Cell Mechanosensitivity

    NASA Astrophysics Data System (ADS)

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

    The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Bone not only develops as a structure designed specifically for mechanical tasks, but it can adapt during life toward more efficient mechanical performance. Mechanical adaptation of bone is a cellular process and needs a biological system that senses the mechanical loading. The loading information must then be communicated to the effector cells that form new bone or destroy old bone.The in vivo operating cell stress derived from bone loading is likely flow of interstitial fluid along the surface of osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin (PG) synthesis and expression of prostaglandin G/H synthase inducible cyclooxygenase (COX-2). Cultured bone cells also rapidly produce nitric oxide (NO) in response to fluid flow as a result of activation of endothelial nitric oxide synthase (ecNOS), which enzyme also mediates the adaptive response of bone tissue to mechanical loading. Disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction.Microgravity, or better near weightlessness, has catabolic effects on the skeleton of astronauts, and on mineral metabolism in bone organ cultures. This might be explained as resulting from an exceptional form of disuse under near weightlessness conditions. However, under near weightlessness conditions the assembly of cytoskeletal elements may be altered since it has been shown that the direction of the gravity vector determines microtubular pattern formation in vivo. We found that the transduction of mechanical signals in bone cells also involves the cytoskeleton and is related to PGE2 production. Therefore it is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions

  19. Collagen-containing scaffolds enhance attachment and proliferation of non-cultured bone marrow multipotential stromal cells.

    PubMed

    El-Jawhari, Jehan J; Sanjurjo-Rodríguez, Clara; Jones, Elena; Giannoudis, Peter V

    2016-04-01

    Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen-containing bovine bone scaffold (Orthoss(®) Collagen) with a non-collagen-containing bovine bone scaffold, Orthoss(®) . Another collagen-containing synthetic scaffold, Vitoss(®) was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit-fibroblast assay and flow-cytometry. The number of BM MSCs initially attached to Orthoss(®) Collagen and Vitoss(®) was similar but greater than Orthoss(®) (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss(®) Collagen and Vitoss(®) after 2-week culture was also higher compared to Orthoss(®) (p = 0.010 and p = 0.023, respectively). Interestingly, collagen-containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture-expanded MSCs on Orthoss(®) collagen and Vitoss(®) was greater compared to Orthoss(®) (p = 0.047 and p = 0.004, respectively). Collectively, collagen-containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes.

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

    PubMed

    Weir, Michael D; Xu, Hockin H K

    2010-04-01

    Because of its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. The objectives of this study were to investigate the response of human mesenchymal stem cells (hMSCs) to a high-strength CPC-chitosan scaffold and to examine cell proliferation and osteogenic differentiation. hMSCs were seeded onto CPC-chitosan composite, CPC control, and tissue culture polystyrene (TCPS). Alkaline phosphatase activity (ALP) and mineralization of hMSCs were measured. CPC-chitosan had a flexural strength (mean + or - SD; n = 5) of (19.5 + or - 1.4) MPa, higher than (8.0 + or - 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 + or - 1.3)% at 8 days, matching (90.7 + or - 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 + or - 11)% at 1 day to (90 + or - 4)% at 8 days (p < 0.05), matching those of CPC control (p > 0.1). Hence, the CPC strength was significantly increased via chitosan without compromising the hMSC response. At 8 days, there was a significant increase in ALP of cells in osteogenic media (10.99 + or - 0.93) [(mM pNpp/min)/(microg DNA)] versus control media (3.62 + or - 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 + or - 19.7)% compared with (6.1 + or - 2.3)% in control medium on TCPS (p < 0.05). In conclusion, hMSCs showed excellent attachment and viability on the strong and tough CPC-chitosan scaffold, matching the hMSC response on CPC control. hMSCs were successfully differentiated down the osteogenic lineage. Hence, the strong, in situ hardening CPC-chitosan scaffold may be useful as a moderate load-bearing vehicle to deliver hMSCs for maxillofacial and orthopedic bone tissue engineering. PMID:20091907

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

    SciTech Connect

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

    2010-09-17

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

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

    NASA Technical Reports Server (NTRS)

    Lawless, Brother Desales

    1990-01-01

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

  3. Collagen‐containing scaffolds enhance attachment and proliferation of non‐cultured bone marrow multipotential stromal cells

    PubMed Central

    El‐Jawhari, Jehan J.; Sanjurjo‐Rodríguez, Clara; Jones, Elena

    2015-01-01

    ABSTRACT Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss®. Another collagen‐containing synthetic scaffold, Vitoss® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss® Collagen and Vitoss® was similar but greater than Orthoss® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss® Collagen and Vitoss® after 2‐week culture was also higher compared to Orthoss® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss® collagen and Vitoss® was greater compared to Orthoss® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016. PMID:26466765

  4. Mouse bone marrow-derived mast cells (BMMC) change their phenotype when cultured with fibroblasts

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Stevens, R.L.

    1986-03-05

    The heparin-containing mast cells (HP-MC) that reside in the connective tissues of the mouse, but not the chondroitin sulfate containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse BMMC (the presumptive in vitro counterpart of the in vivo differentiated mucosal mast cell) were cultured for 2-14 days with confluent skin-derived 3T3 fibroblasts in RPMI-1640 containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin-3 in the conditioned medium. The mast cells remained viable throughout the period of co-culture, since they failed to release LDG and because they increased their histamine content per cell approx.15-fold. After 8-9 days of co-culture, >50% of the BMMC changed histochemically becoming safranin positive. At this time, 30-50% of the (/sup 35/S)glycosaminoglycans on the proteoglycans synthesized by these co-cultured mass cells were heparin, whereas the initial BMMC synthesized proteoglycans containing only chondroitin sulfate E. That interleukin 3-dependent mouse BMMC can be induced to undergo a phenotypic change so as to express characteristics of a HP-MC suggests that the tissue microenvironment determines the differentiated characteristics of these cells.

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

    PubMed

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

    2014-09-01

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

  6. High‐Resolution Three‐Dimensional Computed Tomography Analysis of the Clinical Efficacy of Cultured Autogenous Periosteal Cells in Sinus Lift Bone Grafting

    PubMed Central

    Ogawa, Shin; Hoshina, Hideyuki; Nakata, Koh; Yamada, Kazuho; Uematsu, Kohya; Kawase, Tomoyuki; Takagi, Ritsuo

    2015-01-01

    Abstract Background and Purpose Sinus lift (SL) using cultured autogenous periosteal cells (CAPCs) combined with autogenous bone and platelet‐rich plasma (PRP) was performed to evaluate the effect of cell administration on bone regeneration, by using high‐resolution three‐dimensional computed tomography (CT). Materials and Methods SL with autogenous bone and PRP plus CAPC [CAPC(+)SL] was performed in 23 patients. A piece of periosteum taken from the mandible was cultured in M199 medium with 10% fetal bovine serum (FBS) for 6 weeks. As control, 16 patients received SL with autogenous bone and PRP [CAPC(−)SL]. Three‐dimensional CT imaging was performed before and 4 months and 1 year after SL, and stratification was performed based on CT numbers (HUs) corresponding to soft tissue and cancellous or cortical bone. Results The augmented bone in CAPC(+)SL revealed an increase in HUs corresponding to cancellous bone as well as a decrease in HUs corresponding to grafted cortical bone. In addition, HUs corresponding to cancellous bone in the graft bed were increased in CAPC(+)SL but were decreased in CAPC(−)SL. Insertion torque during implant placement was significantly higher in CAPC(+)SL. Conclusion By promoting bone anabolic activity both in augmented bone and graft bed, CAPCs are expected to aid primary fixation and osseointegration of implants in clinical applications. PMID:26017402

  7. Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration.

    PubMed

    Rossi, M I D; Barros, A P D N; Baptista, L S; Garzoni, L R; Meirelles, M N; Takiya, C M; Pascarelli, B M O; Dutra, H S; Borojevic, R

    2005-10-01

    Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

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

    PubMed Central

    2012-01-01

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

  9. Microgravity and bone cell mechanosensitivity.

    PubMed

    Burger, E H; Klein-Nulend, J

    1998-05-01

    Bone cells, in particular osteocytes, are extremely sensitive to mechanical stress, a quality that is probably linked to the process of mechanical adaptation (Wolff's law). The in vivo operating cell stress derived from bone loading is likely a flow of an interstitial fluid along the surface of the osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin synthesis and expression of inducible prostaglandin G/H synthase (PGHS-2 or inducible cyclooxygenase, COX-2), an enzyme that mediates the induction of bone formation by mechanical loading in vivo. Disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction. Microgravity has catabolic effects on the skeleton of astronauts, as well as on mineral metabolism in bone organ cultures. This might be explained simply as resulting from an exceptional form of disuse under weightlessness conditions. However, under microgravity conditions, the assembly of cytoskeletal elements may be altered, as gravity has been shown to determine the pattern of microtubular orientation assembled in vitro. Therefore, it is possible that the mechanosensitivity of bone cells is altered under microgravity conditions, and that this abnormal mechanosensation contributes to the disturbed bone metabolism observed in astronauts. In vitro experiments on the International Space Station should test this hypothesis experimentally.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

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

    PubMed Central

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

    2014-01-01

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

  12. 3D cell culture and osteogenic differentiation of human bone marrow stromal cells plated onto jet-sprayed or electrospun micro-fiber scaffolds.

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2016-09-01

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

  14. Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic.

    PubMed

    Bara, Jennifer J; Richards, R Geoff; Alini, Mauro; Stoddart, Martin J

    2014-07-01

    Mesenchymal stem cells (MSCs) are increasingly being used in tissue engineering and cell-based therapies in all fields ranging from orthopedic to cardiovascular medicine. Despite years of research and numerous clinical trials, MSC therapies are still very much in development and not considered mainstream treatments. The majority of approaches rely on an in vitro cell expansion phase in monolayer to produce large cell numbers prior to implantation. It is clear from the literature that this in vitro expansion phase causes dramatic changes in MSC phenotype which has very significant implications for the development of effective therapies. Previous reviews have sought to better characterize these cells in their native and in vitro environments, described known stem cell interactions within the bone marrow, and discussed the use of innovative culture systems aiming to model the bone marrow stem cell niche. The purpose of this review is to provide an update on our knowledge of MSCs in their native environment, focusing on bone marrow-derived MSCs. We provide a detailed description of the differences between naive cells and those that have been cultured in vitro and examine the effect of isolation and culture parameters on these phenotypic changes. We explore the concept of "one step" MSC therapy and discuss the potential cellular and clinical benefits. Finally, we describe recent work attempting to model the MSC bone marrow niche, with focus on both basic research and clinical applications and consider the challenges associated with these new generation culture systems.

  15. Research of osteoblastic induced rat bone marrow mesenchymal stem cells cultured on β-TCP/PLLA porous scaffold

    PubMed Central

    Yang, Yi; Wu, Jiang; Jin, Gele; Li, Liang; Li, Zhongwei; Li, Cao

    2015-01-01

    Background: Ceramic and polymer composite scaffolds are widely used in tissue engineering for bone tissue regeneration. Composite of β-tricalcium phosphate (β-TCP) and poly L-lactic acid (PLLA), due to its biocompatibility and biodegradability, is widely used in bioengineering. However, optimal ratio, porosity and pore size of this kind of scaffolds were not very clear yet. Materials and methods: We cultured osteoblastic induced rMSCs on β-TCP/PLLA scaffolds to investigate the optimum construction, which owned better properties for supporting cells growth, proliferation and differentiation. A total of 24 mice were divided into three groups: rMSCs + β-TCP/PLLA, osteoblastic rMSCs + β-TCP/PLLA and β-TCP/PLLA without cells. 8 rude mice were implanted with rMSCs + β-TCP/PLLA in the left thighs and β-TCP/PLLA without cells in the right thighs. 8 rude mice were implanted with osteoblastic rMSCs + β-TCP/PLLA in the left thighs and the same treatments in the right thighs as the above. After 8 and 12 weeks, the mice were sacrificed and implants with the surrounding tissues were harvested together. Paraffin sections were got and HE stain and Masson-Goldner stain were employed to observe the ectopic bone formation. Results: The scaffolds of β-TCP/PLLA = 2:1 significantly increased osteocalcin production of the cells. In addition, scaffolds with NaCl = 70 wt%, pore size 200~450 μm showed better compatibility to these seeding cells. A significantly larger area of bone formation in the osteoblastic rMSCs and β-TCP/PLLA composite than that in rMSCs/scaffold and in the scaffold without cells in vivo. Conclusion: compounds of osteoblastic induced rMSCs and the scaffold with β-TCP/PLLA = 2:1, NaCl = 70 wt%, pore size = 200-450 μm had good properties as a kind of bone substitute. PMID:26064209

  16. Production of calcification inhibitors by rat calvaria and bone cells in culture.

    PubMed

    Ohya, K; Meyer, J L; Felix, R; Fleisch, H

    1988-07-01

    Inhibition of calcium phosphate crystal formation was assessed in conditioned medium from cultured rat calvaria and rat calvaria cells. The amount of inhibitor activity was measured by determining the amount of hydroxyapatite needed to induce calcium phosphate precipitation in a solution with a constant calcium phosphate supersaturation. Rat calvaria in culture released inhibitor activity. This activity was separated by chromatography on a Bio-Gel P4 column into a high molecular weight (HMW) fraction and a low molecular weight (LMW) fraction. The latter contained, among others, citrate and pyrophosphate (PPi), but the concentration of these was too low to account for the total activity observed. The identity of the HMW and the remaining LMW inhibitors are at present unknown. Various populations of calvaria cells also produced these two types of inhibitors, 20% of the LMW being due to PPi. Fibroblasts produced only the HMW type of inhibitors. These results suggest that cells might control the process of biologic calcification by regulating the amount of inhibitors they produce.

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

    PubMed

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

    2015-01-01

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

  18. Inhibition of the insulin-like growth factor-1 receptor enhances effects of simvastatin on prostate cancer cells in co-culture with bone.

    PubMed

    Nordstrand, Annika; Lundholm, Marie; Larsson, Andreas; Lerner, Ulf H; Widmark, Anders; Wikström, Pernilla

    2013-12-01

    Prostate cancer (PC) bone metastases show weak responses to conventional therapies. Bone matrix is rich in growth factors, with insulin-like growth factor-1 (IGF-1) being one of the most abundant. IGF-1 acts as a survival factor for tumor cells and we speculate that bone-derived IGF-1 counteracts effects of therapies aimed to target bone metastases and, consequently, that therapeutic effects could be enhanced if given in combination with IGF-1 receptor (IGF-1R) inhibitors. Simvastatin inhibits the mevalonate pathway and has been found to induce apoptosis of PC cells. The aims of this study were to confirm stimulating effects of bone-derived IGF-1 on PC cells and to test if IGF-1R inhibition enhances growth inhibitory effects of simvastatin on PC cells in a bone microenvironment. The PC-3 and 22Rv1 tumor cell lines showed significantly induced cell growth when co-cultured with neonatal mouse calvarial bones. The tumor cell IGF-1R was activated by calvariae-conditioned media and neutralization of bone-derived IGF-1 abolished the calvarium-induced PC-3 cell growth. Treatment of PC-3 and 22Rv1 cells with simvastatin, or the IGF-1R inhibitor NVP-AEW541, reduced tumor cell numbers and viability, and induced apoptosis. Combined simvastatin and NVP-AEW541 treatment resulted in enhanced growth inhibitory effects compared to either drug given alone. Effects of simvastatin involved down-regulation of IGF-1R in PC-3 and of constitutively active androgen receptor variants in 22Rv1 cells. In conclusion, we suggest that IGF-1 inhibition may be a way to strengthen effects of apoptosis-inducing therapies on PC bone metastases; a possibility that needs to be further tested in pre-clinical models.

  19. Culture and Identification of Mouse Bone Marrow-Derived Dendritic Cells and Their Capability to Induce T Lymphocyte Proliferation

    PubMed Central

    Wang, Wenguang; Li, Jia; Wu, Kun; Azhati, Baihetiya; Rexiati, Mulati

    2016-01-01

    Background The aim of this study was to establish a culture method for mouse dendritic cells (DCs) in vitro and observe their morphology at different growth stages and their ability to induce the proliferation of T lymphocytes. Material/Methods Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were used in combination to induce differentiation of mouse bone marrow (BM) mononucleocytes into DCs. The derived DCs were then assessed for morphology, phenotype, and function. Results The mouse BM-derived mononucleocytes had altered cell morphology 3 days after induction by GM-CSF and IL-4 and grew into colonies. Typical dendrites appeared 8 days after induction. Many mature DCs were generated, with typical dendritic morphology observed under scanning electron microscopy. Expression levels of CD11c, a specific marker of BM-derived DCs, and of co-stimulatory molecules such as CD40, CD80, CD86, and MHC-II were elevated in the mature DCs. Furthermore, the mature DCs displayed a strong potency in stimulating the proliferation of syngenic or allogenic T lymphocytes. Conclusions Mouse BM-derived mononucleocytes cultured in vitro can produce a large number of DCs, as well as immature DCs, in high purity. The described in vitro culture method lays a foundation for further investigations of anti-tumor vaccines. PMID:26802068

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

    SciTech Connect

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

    1991-03-01

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

  1. Kinetics of the appearance of Fc epsilon RI-bearing cells in interleukin-3-dependent mouse bone marrow cultures: correlation with histamine content and mast cell maturation.

    PubMed

    Rottem, M; Barbieri, S; Kinet, J P; Metcalfe, D D

    1992-02-15

    While it is known that mast cells arise from pluripotential hematopoietic cells and express their mature phenotypes in tissues, the sequence of events in maturation is incompletely understood. To study early mast cells, we sorted cells from interleukin-3 (IL-3)-dependent mouse bone marrow cultures on the basis of Fc epsilon RI and examined their morphology, histamine content, and growth characteristics. Flow cytometric analysis and sort showed that the Fc epsilon RI-bearing (Fc epsilon RI+) cells increased from 0% on day 0 to 90% by day 21 and that the total number of Fc epsilon RI+ cells increased from 0 at the start of culture to 3.75 x 10(5) cells by day 21 from an initial population of 1 x 10(5) cells. The dissociation rate of 125I-labeled IgE from early cultured cells resembled the dissociation rate of mouse IgE from mature murine mast cells. Mean fluorescence intensity increased over time, reflecting an increase in IgE receptor density. Fc epsilon RI+ cells were also positive for Fc gamma RII/III. Morphologic studies showed gradual acquisition of metachromatic granules in the Fc epsilon RI+ cells, which was paralleled by an increase in histamine content. Sorted Fc epsilon RI+ cells, when placed in liquid suspension culture, gave rise to pure mast cell populations. Fc epsilon RI+ cells sorted at day 3 and cultured in agarose with IL-3 gave rise to 4,800 small and 150 medium-size mast cell colony-forming units per 10(6) cells, while Fc epsilon RI- cells gave rise to 23 medium-size and 49 large mast cell colony-forming units per 10(6) cells. Fc epsilon RI+ cells grown in granulocyte-macrophage colony-stimulating factor (CSF) or macrophage-CSF did not give rise to colony-forming units. These results show that Fc epsilon RI+ cells have proliferative potential, but that there also is a population of mast cell progenitor cells that have not yet expressed Fc epsilon RI, and such individual progenitor cells have greater potential for proliferation than cells that

  2. Investigation of magnesium-zinc-calcium alloys and bone marrow derived mesenchymal stem cell response in direct culture.

    PubMed

    Cipriano, Aaron F; Sallee, Amy; Guan, Ren-Guo; Zhao, Zhan-Yong; Tayoba, Myla; Sanchez, Jorge; Liu, Huinan

    2015-01-01

    Crystalline Mg-Zn-Ca ternary alloys have recently attracted significant interest for biomedical implant applications due to their promising biocompatibility, bioactivity, biodegradability and mechanical properties. The objective of this study was to characterize as-cast Mg-xZn-0.5Ca (x=0.5, 1.0, 2.0, 4.0wt.%) alloys, and determine the adhesion and morphology of bone marrow derived mesenchymal stem cells (BMSCs) at the interface with the Mg-xZn-0.5Ca alloys. The direct culture method (i.e. seeding cells directly onto the surface of the sample) was established in this study to probe the highly dynamic cell-substrate interface and thus to elucidate the mechanisms of BMSC responses to dynamic alloy degradation. The results showed that the BMSC adhesion density on these alloys was similar to the cell-only positive control and the BMSC morphology appeared more anisotropic on the rapidly degrading alloy surfaces in comparison with the cell-only positive control. Importantly, neither culture media supplemented with up to 27.6mM Mg(2+) ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on BMSC responses. We speculated that degradation-induced dynamic surface topography played an important role in modulating cell morphology at the interface. This study presents a clinically relevant in vitro model for screening bioresorbable alloys, and provides useful design guidelines for determining the degradation rate of implants made of Mg-Zn-Ca alloys.

  3. Models of ex vivo explant cultures: applications in bone research.

    PubMed

    Marino, Silvia; Staines, Katherine Ann; Brown, Genevieve; Howard-Jones, Rachel Anne; Adamczyk, Magdalena

    2016-01-01

    Ex vivo explant culture models are powerful tools in bone research. They allow investigation of bone and cartilage responses to specific stimuli in a controlled manner that closely mimics the in vivo processes. Because of limitations in obtaining healthy human bone samples the explant growth of animal tissue serves as a platform to study the complex physico-chemical properties of the bone. Moreover, these models enable preserving important cell-cell and cell-matrix interactions in order to better understand the behaviour of cells in their natural three-dimensional environment. Thus, the use of bone ex vivo explant cultures can frequently be of more physiological relevance than the use of two-dimensional primary cells grown in vitro. Here, we describe isolation and ex vivo growth of different animal bone explant models including metatarsals, femoral heads, calvaria, mandibular slices and trabecular cores. We also describe how these explants are utilised to study bone development, cartilage and bone metabolism, cancer-induced bone diseases, stem cell-driven bone repair and mechanoadaptation. These techniques can be directly used to understand mechanisms linked with bone physiology or bone-associated diseases.

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    1996-01-01

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

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

    PubMed

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

    2013-02-01

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

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

    PubMed

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

    1991-05-01

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

  8. Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures.

    PubMed

    de Oliveira, Paulo Tambasco; de Oliva, Marcos Andrade; Maximiano, William Marcatti Amarú; Sebastião, Karen Elaine Vasconcelos; Crippa, Grasiele Edilaine; Ciancaglini, Pietro; Beloti, Márcio Mateus; Nanci, Antonio; Rosa, Adalberto Luiz

    2008-07-01

    Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.

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

    SciTech Connect

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

    1988-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  13. [Cell cultures].

    PubMed

    Cipro, Simon; Groh, Tomáš

    2014-01-01

    Cell or tissue cultures (both terms are interchangeable) represent a complex process by which eukaryotic cells are maintained in vitro outside their natural environment. They have a broad usage covering not only scientific field but also diagnostic one since they represent the most important way of monoclonal antibodies production which are used for both diagnostic and therapeutic purposes. Cell cultures are also used as a "cultivation medium" in virology and for establishing proliferating cells in cytodiagnostics. They are well-established and easy-to-handle models in the area of research, e.g. as a precious source of nucleic acids or proteins. This paper briefly summarizes their importance and methods as well as the pitfalls of the cultivation and new trends in this field. PMID:24624984

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

    PubMed Central

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

    2004-01-01

    Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture. PMID:15194807

  15. Bone-immune cell crosstalk: bone diseases.

    PubMed

    Mori, Giorgio; D'Amelio, Patrizia; Faccio, Roberta; Brunetti, Giacomina

    2015-01-01

    Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma. PMID:26000310

  16. Bone-immune cell crosstalk: bone diseases.

    PubMed

    Mori, Giorgio; D'Amelio, Patrizia; Faccio, Roberta; Brunetti, Giacomina

    2015-01-01

    Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma.

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

    PubMed

    Stains, Joseph P; Civitelli, Roberto

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    SciTech Connect

    Greenberger, J.S.; Eckner, R.J.; Otten, J.A.; Tennant, R.W.

    1982-07-01

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

  20. Wnt5a is expressed in spondyloarthritis and exerts opposite effects on enthesis and bone in murine organ and cell cultures.

    PubMed

    Bougault, Carole; Briolay, Anne; Boutet, Marie-Astrid; Pilet, Paul; Delplace, Séverine; Le Goff, Benoît; Guicheux, Jérôme; Blanchard, Frédéric; Magne, David

    2015-12-01

    Spondyloarthritis (SpA) is a chronic inflammatory joint disorder that initiates at the enthesis, where tendons attach to bone through a fibrocartilage zone. At late stages, excessive bone apposition appears within the diseased enthesis. Because Wnt5a participates to normal bone formation and appears related to inflammatory processes, we investigated the role of this Wnt growth factor in inflammation-associated ossification in SpA. The concentration of Wnt5a assessed by enzyme-linked immunosorbent assay in synovial fluids of patients with SpA (2.58 ± 0.98 ng/mL) was higher than in osteoarthritic patients (1.33 ± 0.71 ng/mL). In murine primary cultures of tendon cells, chondrocytes, and osteoblasts and in an organotypic model of mouse ankle, we showed that tumor necrosis factor α reversibly diminished Wnt5a expression and secretion, respectively. Wnt5a decreased gene expression of differentiation markers and mineralization in cultured chondrocytes and reduced alkaline phosphatase activity in Achilles tendon enthesis (-14%) and osteocalcin protein levels released by ankle explants (-36%). On the contrary, Wnt5a stimulated ossification markers' expression in cultured osteoblasts and increased the bone volume of the tibial plateau of the cultured explants (+19%). In conclusion, our results suggest that Wnt5a is expressed locally in the joints of patients with SpA. Wnt5a appears more associated with ossification than with inflammation and tends to inhibit mineralization in chondrocytes and enthesis, whereas it seems to favor the ossification process in osteoblasts and bone. Further studies are needed to decipher the opposing effects observed locally in enthesis and systemically in bone in SpA.

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    1980-08-01

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

  5. A Comparison of Three-Dimensional Culture Systems to Evaluate In Vitro Chondrogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Watts, Ashlee E.; Ackerman-Yost, Jeremy C.

    2013-01-01

    Objective To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods MSCs from five horses 2–3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×105 cell pellets, 5×105 cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X). Results alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5×105 cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels. Conclusion equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems. PMID:23725547

  6. Bone cell proliferation on carbon nanotubes.

    PubMed

    Zanello, Laura P; Zhao, Bin; Hu, Hui; Haddon, Robert C

    2006-03-01

    We explored the use of carbon nanotubes (CNTs) as suitable scaffold materials for osteoblast proliferation and bone formation. With the aim of controlling cell growth, osteosarcoma ROS 17/2.8 cells were cultured on chemically modified single-walled (SW) and multiwalled (MW) CNTs. CNTs carrying neutral electric charge sustained the highest cell growth and production of plate-shaped crystals. There was a dramatic change in cell morphology in osteoblasts cultured on MWNTs, which correlated with changes in plasma membrane functions.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2012-10-01

    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.

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

    SciTech Connect

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

    2012-05-25

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    1985-04-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

    Diabetic periodontitis (DP), which has been shown to cause alveolar bone loss, is among the most common complications associated with diabetes. The precise mechanisms underlying alveolar bone loss in patients with DP remain unclear. Therefore, the present study established a co-culture system of bone marrow stem cells (BMSCs) and macrophages, in order to investigate the potential mechanisms underlying DP-associated alveolar bone loss in vitro. In addition, Porphyromonas gingivalis (PG) periodontal infection and high glucose levels were used to induce DP in mice. The present study evaluated the protein expression levels of various chemokines and the migration of BMSCs and macrophages. The protein expression levels of extracellular signal-regulated kinase 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) were significantly increased in the BMSCs exposed to high glucose and PG, which may have been due to the activation of MAPK. In addition, DP induction in mice was associated with the release of chemokine (C-C motif) ligand 2 (CCL2) from BMSCs and the secretion of chemokine (C-C Motif) receptor 2 (CCR2) and tumor necrosis factor-α from macrophages, which was associated in turn with enhanced adhesion and chemotaxis of macrophages. The results of the present study suggested that DP led to the upregulation of CCL2 in the periodontal tissues and enhanced macrophage infiltration via the CCL2/CCR2 axis, which in turn promoted alveolar bone loss. PMID:27446245

  14. Clinical utility of bone marrow culture.

    PubMed

    Moore, M A

    1976-01-01

    Standardized culture of bone marrow in soft agar permits the detection of a population of granulocyte-macrophage progenitor cells (CFU-c). A spectrum of qualitative abnormalities serves to distinguish myeloid leukemic CFU-c from normal and remission populations. These abnormalities in maturation and proliferation are diagnostic of a myeloid leukemic state and serve to functionally reclassify acute myeloid leukemia at diagnosis into a number of categories based on in vitro growth pattern. The virtue of this classification is that it permits detection of a substantial number of patients who are refractory to conventional remission induction protocols. The clear distinction between normal and leukemic growth in vitro permits early detection of emerging remission CFU-c during induction therapy and of early onset of relapse in patients who are otherwise in complete remission. In patients with leukemia undergoing allogeneic bone marrow engraftment, marrow culture has proved of value in documenting the reconstitution of the patient and in detecting re-emergence of the original leukemic stem line prior to its detection by cytogenetic and hematological techniques. Serial studies on patients with chronic myeloid leukemia have allowed early diagnosis of blastic transformation and classification of blastic phase disease on the basis of in vitro growth pattern has revealed a similar spectrum of in vitro abnormalities as seen in AML. The cloning of normal or leukemic human myeloid progenitor cells (CFU-c) in agar or methylcellulose has permitted analysis of both quantitative and qualitative changes in this cell compartment in leukemia and other myelodysplastic states (1-7). Among these changes are abnormalities in maturation of leukemic cells in vitro (4, 5, 6), defective proliferation as measured by colony size or cluster to colony ratio (5, 6), abnormalities in biophysical characteristics of leukemic CFU-c (4, 5), regulatory defects in responsiveness to positive and negative

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

    PubMed Central

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

    1985-01-01

    S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR 2721) is a radio- and chemoprotective agent which produces hypocalcemia in humans. Intravenous injection of 30 mg/kg WR 2721 in rats and 15 mg/kg in dogs lowers serum calcium by 19 and 25%, respectively. Hypocalcemia in dogs is associated with a fall in serum immunoreactive parathyroid hormone (PTH), which suggests that the mechanism of its hypocalcemic effect is acute hypoparathyroidism. Despite this effect on PTH, in eight chronically parathyroidectomized rats on a low phosphate diet, WR 2721 reduced serum calcium from 9.4 +/- 0.6 to 7.7 +/- 0.5 mg/dl (P less than 0.01) at 3 h. Furthermore, in dogs rendered hypercalcemic by continuous infusion of PTH, WR 2721 reduced serum calcium from 11.0 +/- 0.5 to 10.6 +/- 0.5 mg/dl (P less than 0.01). To determine whether WR 2721 causes hypocalcemia by enhancing the exit of calcium from the circulation or inhibiting its entry, the drug was infused 3 h after administration of 45Ca to rats. WR 2721 did not significantly increase the rate of disappearance of 45Ca from the circulation even though serum calcium fell by 19%. However, serum 45Ca specific activity was higher at 1.5 h (P less than 0.01) and 3 h (P less than 0.05) in rats given WR 2721 than in rats given vehicle alone, which suggests that WR 2721 blocks the entry of nonradioactive calcium into the circulation, presumably from bone. In incubations with fetal rat long bone labeled in utero with 45Ca, 10(-3) M WR 2721 inhibited PTH-stimulated, but not base-line release of 45Ca. Bone resorption by primary culture of chick osteoclasts was inhibited by WR 2721 at concentrations as low as 10(-4) M in the absence of hormonal stimulation. These studies suggest that WR 2721 lowers serum calcium predominantly by blocking calcium release from bone. This acute hypocalcemic effect is at least in part independent of its effect on the parathyroid glands, and is most likely a direct effect of the agent on bone resorption. PMID

  16. A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.

    PubMed

    Carmelo, Joana G; Fernandes-Platzgummer, Ana; Diogo, Maria Margarida; da Silva, Cláudia Lobato; Cabral, Joaquim M S

    2015-08-01

    Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield. Maximal cell densities of 3.6 × 10(5) and 1.9 × 10(5) cells/mL were obtained for BM MSC (0.60 ± 0.04 day(-1) ) and ASC (0.9 ± 0.1 day(-1) ) cultures, upon seven and eight days of cultivation, respectively. Ready-to-use microcarriers Synthemax® II and Enhanced Attachment® supported identical expansion performance of BM MSC, turning those effective alternatives to the pre-coated plastic microcarriers used in our xeno-free scalable culture system. Importantly, expanded MSC maintained their immunophenotype and multilineage differentiation potential. Moreover, secretome analysis suggested a priming effect of stirred culture conditions on cytokine production by MSC. This culture system yielded considerable final cell densities that can be scaled-up to controlled large-scale bioreactors allowing a more efficient, safe and cost-effective MSC production for clinical settings.

  17. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

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

  18. Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum

    PubMed Central

    Esmaeli, Azadeh; Moshrefi, Mojgan; Shamsara, Ali; Eftekhar-vaghefi, Seyed Hasan; Nematollahi-mahani, Seyed Noureddin

    2016-01-01

    Background: Fetal bovine serum (FBS) is widely used in cell culture laboratories, risk of zoonotic infections and allergic side effects create obstacles for its use in clinical trials. Therefore, an alternative supplement with proper inherent growth-promoting activities is demanded. Objective: To find FBS substitute, we tested human umbilical cord blood serum (hUCS) for proliferation of human umbilical cord matrix derived mesenchymal stem cells (hUC-MSCs) and human bone marrow-derived mesenchymal cells (hBM-MSCs). Materials and Methods: Umbilical cord blood of healthy neonates, delivered by Caesarian section, was collected and the serum was separated. hUC-MSCs and hBM-MSCs were isolated and characterized by assessment of cell surface antigens by flow cytometry, alkaline phosphatase activity and osteogenic/adipogenic differentiation potential. The cells were then cultured in Iscove's Modified Dulbecco's Medium (IMDM) by conventional methods in three preparations: 1- with hUCS, 2- with FBS, and 3- without serum supplements. Cell proliferation was measured using WST-1 assay, and cell viability was assessed by trypan blue staining. Results: The cells cultured in hUCS and FBS exhibited similar morphology and mesenchymal stem cells properties. WST-1 proliferation assay data showed no significant difference between the proliferation rate of either cells following hUCS and FBS supplementation. Trypan blue exclusion dye test also revealed no significant difference for viability between hUCS and FBS groups. A significant difference was detected between the proliferation rate of stem cells cultured in serum-supplemented medium compared with serum-free medium. Conclusion: Our results indicate that human umbilical cord serum can effectively support proliferation of hBM-MSCS and hUC-MSCs in vitro and can be used as an appropriate substitute for FBS, especially in clinical studies. PMID:27738658

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2010-04-01

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

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

    PubMed

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

    2010-04-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. [Cytokine profile of a human bone marrow cell culture under the influence of UHMW-PE wear particles].

    PubMed

    Wilke, A; Bartsch, I; Kratz, M; Jones, D; Endres, S

    2005-10-01

    There is considerable evidence that orthopaedic wear debris plays a crucial role in the pathology of aseptic loosening of joint prostheses. The purpose of the present study was to evaluate the influence of ultra-high-molecular-weight polyethylene (UHMW-PE) on the cytokine response in a modified in vitro model. UHMW-PE particles (psi < 7.5 microm) were suspended in soluble collagen type I and subsequently solidified in different concentrations (105,106 and 107 particles per well) on the bottom of the wells. Human bone marrow cells in a concentration of 3 x 106 cells per well were seeded on the collagen-particle substrata and maintained for up to 12 days. The cytokine response (IL-1_, IL-6 and TNF-_) of the cells to the particles were examined by ELISA compared to cells on control collagen surfaces without any particles. Assays for viability using LDH activity were done immediately. Light and scanning microscopic evaluation revealed that the UHMWPE particles, which have built large conglomerates (psi7.5_m), were mainly surrounded by the cells and less phagocytosed. The results of the cytokine release revealed significant differences in interleukin (IL)6, tumor necrosis factor (TNF)- _ and IL-1beta. The cell viability was not affected by the UHMW-PE particles. The results demonstrate that the particle induced cytokine response by UHMW-PE is mainly by the release of Interleukin 6 and TNF- _. Moreover the results confirm that the present method is useful to evaluate the in vitro effects of UHMW-PE wear particles with direct particle cell contact. PMID:16300048

  5. Clonal T-cell colony formation in agar culture: an attractive assay to test the T-cell depletion from bone marrow.

    PubMed

    Farcet, J P; Beaujean, F; Cordonnier, C; Pico, J; Gourdin, M F; Divine, M; Bracq, C; Bouguet, J; Laurent, G; Bernard, A

    1986-12-01

    Current studies suggest that the depletion of T-lymphocytes from donor marrow is an effective method for preventing acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation in man. To deplete the T-lymphocytes from bone marrow cells we use either monoclonal anti-T-cell antibodies and complement or T101 ricin A-chain immunotoxin. Residual T-lymphocytes are analyzed by their capacity to form clonal T-cell colonies in the presence of phytohemagglutinin (PHA), accessory cells, and recombinant interleukin 2. The method is compared to immediate indirect immunofluorescence (iF) and thymidine incorporation by marrow cells stimulated by PHA. IF is not suitable for evaluating the depletion by immunotoxin, and the interpretation of thymidine incorporation is generally questionable. The results of the colony formation show that the sensitivity of the colony assay is close to that of iF when T cells are depleted by complement lysis, and the sensitivity of the colony assay is not dependent upon the depletion procedure. Therefore, the T-cell colony assay is a simple functional control for the quality of bone marrow T-cell depletion, especially for T-cell depletion by immunotoxin. PMID:3536543

  6. Osteostimulatory effect of bone grafts on fibroblast cultures

    PubMed Central

    Fathima, Hameed; Harish

    2015-01-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-06-01

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

  9. Human bone marrow-derived stromal cells cultured with a plasma sprayed CaO-ZrO2-SiO2 coating.

    PubMed

    Yang, Fei; Xie, Youtao; Li, Huiwu; Tang, Tingting; Zhang, Xiaoling; Gan, Yaokai; Zheng, Xuebin; Dai, Kerong

    2010-10-01

    The CaO-ZrO2-SiO2 (CZS) coating was prepared by plasma-spraying chemically synthesized CZS powder onto a Ti-6Al-4V substrate. This CZS coating has been demonstrated to have good bioactivity, high bonding strength with the substrate and a low degradation rate. However, the effect of CZS coating on the osseointegration of bone-implant is still unknown. In this study, human bone marrow-derived stromal cells (hBMSCs) were cultured on CZS coating in vitro, and cell behavior was investigated, with the classical hydroxyapatite (HA) coating as a control. Scanning electron microscopy (SEM) and immunofluorescence studies showed that the hBMSCs on the CZS coating spread well with organized cytoskeleton structure at 24 h following cell seeding. The MTT assay and the Alamar Blue assay indicated that CZS coating promoted the attachment and proliferation of hBMSCs. The results of alkaline phosphatase (ALP) activity test and the expression of osteogenic marker genes, such as ALP, collagen I (COLI), osteopontin (OPN), and osteocalcin (OCN), demonstrated that the osteoblastic differentiation of hBMSCs was enhanced more by CZS coating than by HA coating. These results suggest that CZS coating possess excellent biological properties and may have potential in biomedical applications.

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

    PubMed

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

    2014-08-01

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

  11. Bone cells-biomaterials interactions.

    PubMed

    Marquis, Marie-Eve; Lord, Etienne; Bergeron, Eric; Drevelle, Olivier; Park, Hyunjin; Cabana, Francois; Senta, Helena; Faucheux, Nathalie

    2009-01-01

    With the aging population, the incidence of bone defects due to fractures, tumors and infection will increase. Therefore, bone replacement will become an ever bigger and more costly problem. The current standard for bone replacement is autograft, because these transplants are osteoconductive and osteoinductive. However, harvesting an autograft requires additional surgery at the donor site that is related to high level of morbidity. In addition, the quantity of bone tissue that can be harvested is limited. These limitations have necessitated the pursuit of alternatives using biomaterials. The control of bone tissue cell adhesion to biomaterials is an important requirement for the successful incorporation of implants or the colonization of scaffolds for tissue repair. Controlling cells-biomaterials interactions appears of prime importance to influence subsequent biological processes such as cell proliferation and differentiation. Therefore, interactions of cells with biomaterials have been widely studied especially on two-dimensional systems. This review focuses on these interactions.

  12. Uptake of a fluorinated bisphosphonate by cultured bones

    SciTech Connect

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

    1988-01-01

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

  13. The effect of timing of mechanical stimulation on proliferation and differentiation of goat bone marrow stem cells cultured on braided PLGA scaffolds.

    PubMed

    van Eijk, Floor; Saris, Daniel B F; Creemers, Laura B; Riesle, Jens; Willems, W Jaap; van Blitterswijk, Clemens A; Verbout, Abraham J; Dhert, Wouter J A

    2008-08-01

    Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.

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

    PubMed

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

    2015-05-01

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

  15. Osteogenic activity of bone marrow-derived mesenchymal stem cells (BMSCs) seeded on irradiated allogenic bone.

    PubMed

    Tohma, Yasuaki; Dohi, Yoshiko; Ohgushi, Hajime; Tadokoro, Mika; Akahane, Manabu; Tanaka, Yasuhito

    2012-02-01

    Allogenic bone grafting, a technique used in orthopaedic surgery, has several problems, including low osteogenic activity. To overcome the problem, this study aimed to determine whether in vivo osteogenesis could be enhanced using allogenic irradiated bone grafts after seeding with autologous bone marrow-derived mesenchymal stem cells (BMSCs). The allogenic bone cylinders were extracted from ACI rats and sterilized by irradiation. Donor BMSCs were obtained from fresh Fischer 344 (F344) rat bone marrow by cell culture. The allogenic bone with or without BMSCs were transplanted subcutaneously into syngeneic F344 rats. At 4 weeks after transplantation, high alkaline phosphatase (ALP) activity, bone-specific osteocalcin mRNA expression and newly formed bone were detected in the allogenic bone with BMSCs. The origin of the newly formed bone was derived from cultured donor BMSCs. However, none of these identifiers of osteogenesis were detected in either the fresh or the irradiated allogenic bone without BMSCs. These results indicate the availability of autologous BMSCs to heighten the osteogenic response of allogenic bone. Our present tissue-engineering method might contribute to a wide variety of allogenic bone grafting techniques in clinical settings.

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

    PubMed

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

    2015-01-01

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

  17. STROMAS: A Series of Microgravity Experiments on Bone Forming Cells

    NASA Astrophysics Data System (ADS)

    Yi, Liu; Massimilano, Monticone; Federico, Tortelli; Matalija, Pujic; Alessandra, Ruggiu; Ranieri, Cancedda

    2008-06-01

    We developed a novel 3D in vitro culture system by seeding cells onto porous bioceramics, mimicking the physiological niche of bone turn-over and enhancing cellular differentiation respective to conventional 2D Petri Dish cultures. Having overcome several technological difficulties, in a series of STROMA spaceflight experiments 3D cultures of bone marrow derived mesenchymal stem cells (BMSC) and co-cultures of osteoblasts and osteoclast precursors were maintained and conserved in automated bioreactors on orbit. Genechip analysis revealed an inhibition of cell proliferation in microgravity. Unexpectedly, genes related to various processes of neural development were significantly upregulated in microgravity, raising the question on the lineage restriction in BMSC.

  18. Bone regeneration and stem cells

    PubMed Central

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

    2011-01-01

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

  19. [Features of development of fetal bone organ culture in space flight].

    PubMed

    Berezovskaia, O P

    2002-01-01

    Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.

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

    PubMed

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

    2016-08-01

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

  1. Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche

    PubMed Central

    Huang, Xiaobing; Zhu, Biao; Wang, Xiaodong; Xiao, Rong; Wang, Chunsen

    2016-01-01

    Recent studies have indicated that the hematopoietic stem/progenitor cell (HSPC) niche, consisting of two major crucial components, namely osteoblasts (OBs) and mesenchymal stromal cells (MSCs), is responsible for the fate of HSPCs. Thus, closely mimicking the HSPC niche ex vivo may be an efficient strategy with which to develop new culture strategies to specifically regulate the balance between HSPC self-renewal and proliferation. The aim of this study was to establish a novel HSPC three-dimensional culture system by co-culturing bone marrow-derived MSCs and OBs differentiated from MSCs without any cytokines as feeder cells and applying bio-derived bone from human femoral metaphyseal portion as the scaffold. Scanning electron microscopy revealed the excellent biocompatibility of bio-derived bone with bone marrow-derived MSCs and OBs differentiated from MSCs. Western blot analysis revealed that many cytokines, which play key roles in HSPC regulation, were comprehensively secreted, while ELISA revealed that extracellular matrix molecules were also highly expressed. Hoechst 33342/propidium iodide fluorescence staining proved that our system could be used to supply a long-term culture of HSPCs. Flow cytometric analysis and qPCR of p21 expression demonstrated that our system significantly promoted the self-renewal and ex vivo expansion of HSPCs. Colony-forming unit (CFU) and long-term culture-initiating cell (LTC-IC) assays confirmed that our system has the ability for both the expansion of CD34+ hematopoietic stem cells (HPCs) and the maintenance of a primitive cell subpopulation of HSCs. The severe-combined immunodeficient mouse repopulating cell assay revealed the promoting effects of our system on the expansion of long-term primitive transplantable HSCs. In conclusion, our system may be a more comprehensive and balanced system which not only promotes the self-renewal and ex vivo expansion of HSPCs, but also maintains primitive HPCs with superior phenotypic and

  2. 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

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

    1988-08-01

    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.

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

    PubMed

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

    1999-08-01

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

  4. 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.

    PubMed

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

    2012-05-01

    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-α 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  6. In vivo bone regeneration using tubular perfusion system bioreactor cultured nanofibrous scaffolds.

    PubMed

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

    2014-01-01

    The use of bioreactors for the in vitro culture of constructs for bone tissue engineering has become prevalent as these systems may improve the growth and differentiation of a cultured cell population. Here we utilize a tubular perfusion system (TPS) bioreactor for the in vitro culture of human mesenchymal stem cells (hMSCs) and implant the cultured constructs into rat femoral condyle defects. Using nanofibrous electrospun poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffolds, hMSCs were cultured for 10 days in vitro in the TPS bioreactor with cellular and acellular scaffolds cultured statically for 10 days as a control. After 3 and 6 weeks of in vivo culture, explants were removed and subjected to histomorphometric analysis. Results indicated more rapid bone regeneration in defects implanted with bioreactor cultured scaffolds with a new bone area of 1.23 ± 0.35 mm(2) at 21 days compared to 0.99 ± 0.43 mm(2) and 0.50 ± 0.29 mm(2) in defects implanted with statically cultured scaffolds and acellular scaffolds, respectively. At the 21 day timepoint, statistical differences (p<0.05) were only observed between defects implanted with cell containing scaffolds and the acellular control. After 42 days, however, defects implanted with TPS cultured scaffolds had the greatest new bone area with 1.72 ± 0.40 mm(2). Defects implanted with statically cultured and acellular scaffolds had a new bone area of 1.26 ± 0.43 mm(2) and 1.19 ± 0.33 mm(2), respectively. The increase in bone growth observed in defects implanted with TPS cultured scaffolds was statistically significant (p<0.05) when compared to both the static and acellular groups at this timepoint. This study demonstrates the efficacy of the TPS bioreactor to improve bone tissue regeneration and highlights the benefits of utilizing perfusion bioreactor systems to culture MSCs for bone tissue engineering.

  7. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits

    PubMed Central

    LI, PU; ZHANG, LEI

    2015-01-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  8. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.

    PubMed

    Li, Pu; Zhang, Lei

    2015-08-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  9. Cancer Cell Colonisation in the Bone Microenvironment

    PubMed Central

    Kan, Casina; Vargas, Geoffrey; Le Pape, François; Clézardin, Philippe

    2016-01-01

    Bone metastases are a common complication of epithelial cancers, of which breast, prostate and lung carcinomas are the most common. The establishment of cancer cells to distant sites such as the bone microenvironment requires multiple steps. Tumour cells can acquire properties to allow epithelial-to-mesenchymal transition, extravasation and migration. Within the bone metastatic niche, disseminated tumour cells may enter a dormancy stage or proliferate to adapt and survive, interacting with bone cells such as hematopoietic stem cells, osteoblasts and osteoclasts. Cross-talk with the bone may alter tumour cell properties and, conversely, tumour cells may also acquire characteristics of the surrounding microenvironment, in a process known as osteomimicry. Alternatively, these cells may also express osteomimetic genes that allow cell survival or favour seeding to the bone marrow. The seeding of tumour cells in the bone disrupts bone-forming and bone-resorbing activities, which can lead to macrometastasis in bone. At present, bone macrometastases are incurable with only palliative treatment available. A better understanding of how these processes influence the early onset of bone metastasis may give insight into potential therapies. This review will focus on the early steps of bone colonisation, once disseminated tumour cells enter the bone marrow. PMID:27782035

  10. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

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

  11. [Coupling and communication between bone cells].

    PubMed

    Nakashima, Tomoki

    2014-06-01

    Bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic 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. Coupling has been understood as a balanced induction of osteoblastic bone formation in response to osteoclastic bone resorption. An imbalance of this coupling is often linked to various bone diseases. TGF-β and IGF released from bone matrix during osteoclastic bone resorption are the favored candidates as classical coupling factor. Recently, several reports suggest that osteoclast-derived molecules/cytokines (clastokine) mediate directional signaling between osteoblasts and osteoclasts into the bone microenvironment. Thus, the elucidation of the regulatory mechanisms involved in bone cell communication and coupling is critical for a deeper understanding of the skeletal system in health and disease.

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

    SciTech Connect

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

    2011-04-01

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

  13. Mechanotransduction of bone cells in vitro: mechanobiology of bone tissue.

    PubMed

    Mullender, M; El Haj, A J; Yang, Y; van Duin, M A; Burger, E H; Klein-Nulend, J

    2004-01-01

    Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E2 values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6 +/- 0.3 Pa). Cyclic strain (1000 microstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E2. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.

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

    PubMed Central

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

    2015-01-01

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

  15. Organotypic culture of human bone marrow adipose tissue.

    PubMed

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

    2010-04-01

    The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was <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.

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

    PubMed

    Mizuno, Hiroki; Kikuta, Junichi; Ishii, Masaru

    2014-04-01

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

  17. Fabrication and perfusion culture of anatomically shaped artificial bone using stereolithography.

    PubMed

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

    2014-09-12

    Because patient bone defects are usually varied and complicated in geometry, it would be preferred to fabricate custom-made artificial bone grafts that are anatomically specific to individual patient defects. Using a rabbit femoral segment as a bone reconstruction model, we successfully produced customized ceramic scaffolds by stereolithography, which not only had an anatomically correct external shape according to computed tomography data but also contained an interconnecting internal network of channels designed for perfusion culture. Rabbit bone marrow stromal cells were isolated and cultured with these scaffolds using a novel oscillatory perfusion system that was stereolithographically fabricated to fit well to the unique scaffold shapes. After five days of three-dimensional culture with oscillatory perfusion, the cells attached and proliferated homogenously in the scaffolds. However, control cells inside the scaffolds cultured under static conditions were dead after prolonged in vitro culture. Cellular DNA content and alkaline phosphatase activities were significantly higher in the perfusion group versus the static group. Therefore, anatomically correct artificial bone can be successfully constructed using stereolithography and oscillatory culture technology, and could be useful for bone engraftment and defect repair.

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

    PubMed

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

    2015-06-01

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

  19. Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

    PubMed

    Caballé-Serrano, J; Cvikl, B; Bosshardt, D D; Buser, D; Lussi, A; Gruber, R

    2015-01-01

    Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

  20. Bone marrow cells differentiation into organ cells using stem cell therapy.

    PubMed

    Yang, Y-J; Li, X-L; Xue, Y; Zhang, C-X; Wang, Y; Hu, X; Dai, Q

    2016-07-01

    Bone marrow cells (BMC) are progenitors of bone, cartilage, skeletal tissue, the hematopoiesis-supporting stroma and adipocyte cells. BMCs have the potential to differentiate into neural cells, cardiac myocytes, liver hepatocytes, chondrocytes, renal, corneal, blood, and myogenic cells. The bone marrow cell cultures from stromal and mesenchymal cells are called multipotent adult progenitor cells (MAPCs). MAPCs can differentiate into mesenchymal cells, visceral mesoderm, neuroectoderm and endoderm in vitro. It has been shown that the stem cells derived from bone marrow cells (BMCs) can regenerate cardiac myocytes after myocardial infarction (MI). Adult bone marrow mesenchymal stem cells have the ability to regenerate neural cells. Neural stem/progenitor cells (NS/PC) are ideal for treating central nervous system (CNS) diseases, such as Alzheimer's, Parkinson's and Huntington disease. However, there are important ethical issues about the therapeutic use of stem cells. Neurons, cardiac myocytes, hepatocytes, renal cells, blood cells, chondrocytes and adipocytes regeneration from BMCs are very important in disease control. It is known that limbal epithelial stem cells in the cornea can repair the eye sight and remove symptoms of blindness. Stem cell therapy (SCT) is progressing well in animal models, but the use of SCT in human remains to be explored further.

  1. The regulation of bone turnover in ameloblastoma using an organotypic in vitro co-culture model

    PubMed Central

    Eriksson, Tuula M; Day, Richard M; Fedele, Stefano; Salih, Vehid M

    2016-01-01

    Ameloblastoma is a rare, odontogenic neoplasm with benign histopathology, but extensive, local infiltrative capacity through the bone tissue it originates in. While the mechanisms of ameloblastoma invasion through the bone and bone absorption are largely unknown, recent investigations have indicated a role of the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand regulatory mechanisms. Here, we present results obtained using a novel in vitro organotypic tumour model, which we have developed using tissue engineering techniques. Using this model, we analysed the expression of genes involved in bone turnover and detected a 700-fold increase in receptor activator of nuclear factor kappa-B ligand levels in the co-culture models with ameloblastoma cells cultured with bone cells. The model described here can be used for gene expression studies, as a basis for drug testing or for a more tailored platform for testing of the behaviour of different ameloblastoma tumours in vitro. PMID:27746893

  2. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

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

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

    PubMed

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

    2012-04-01

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

  4. Fish stem cell cultures.

    PubMed

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  5. Detection of apoptosis of bone cells in vitro.

    PubMed

    Bellido, Teresita; Plotkin, Lilian I

    2008-01-01

    Studies during the last decade demonstrated that apoptosis is as important as mitosis for the growth and maintenance of the skeleton and provided information on the significance and molecular regulation of apoptosis of bone cells. It is now known that: (1) all osteoclasts die by apoptosis after completing a bone resorption cycle; (2) the majority of osteoblasts also die, whereas the remainder become lining cells or osteocytes; and (3) osteocytes, although long-living cells, also can die prematurely. Furthermore, mounting evidence indicates that systemic hormones, local growth factors, cytokines, and pharmacological agents, as well as mechanical forces regulate the rate of bone cell apoptosis. This chapter summarizes the methods developed in the last few years to examine apoptosis of cultured bone cells and identify the signaling pathways and molecules involved in apoptosis regulation by diverse skeletal stimuli.

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

    PubMed

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

    2014-04-01

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

  7. Reversing bone loss by directing mesenchymal stem cells to bone.

    PubMed

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

    2013-09-01

    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 α4β1 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.

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

    PubMed

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

    2011-10-01

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

  9. Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals.

    PubMed

    Nishimori, Hikaru; Ehata, Shogo; Suzuki, Hiroshi I; Katsuno, Yoko; Miyazono, Kohei

    2012-06-01

    Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.

  10. Digital Microfluidic Cell Culture.

    PubMed

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

    2015-01-01

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

  11. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects.

    PubMed

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

    2012-07-01

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

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

    PubMed

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

    2013-07-01

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

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

    PubMed

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

    2014-09-01

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

  14. Degradation of polysaccharide hydrogels seeded with bone marrow stromal cells.

    PubMed

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

    2011-10-01

    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.

  15. Renal Cell Carcinoma Metastasized to Pagetic Bone

    PubMed Central

    Ramirez, Ashley; Liu, Bo; Rop, Baiywo; Edison, Michelle; Valente, Michael

    2016-01-01

    Paget’s disease of the bone, historically known as osteitis deformans, is an uncommon disease typically affecting individuals of European descent. Patients with Paget’s disease of the bone are at increased risk for primary bone neoplasms, particularly osteosarcoma. Many cases of metastatic disease to pagetic bone have been reported. However, renal cell carcinoma metastasized to pagetic bone is extremely rare. A 94-year-old male presented to the emergency department complaining of abdominal pain. A computed tomography scan of the abdomen demonstrated a large mass in the right kidney compatible with renal cell carcinoma. The patient was also noted to have Paget’s disease of the pelvic bones and sacrum. Within the pagetic bone of the sacrum, there was an enhancing mass compatible with renal cell carcinoma. A subsequent biopsy of the renal lesion confirmed renal cell carcinoma. Paget’s disease of the bone places the patient at an increased risk for bone neoplasms. The most commonly reported sites for malignant transformation are the femur, pelvis, and humerus. In cases of malignant transformation, osteosarcoma is the most common diagnosis. Breast, lung, and prostate carcinomas are the most common to metastasize to pagetic bone. Renal cell carcinoma associated with Paget’s disease of the bone is very rare, with only one prior reported case. Malignancy in Paget's disease of the bone is uncommon with metastatic disease to pagetic bone being extremely rare. We report a patient diagnosed with concomitant renal cell carcinoma and metastatic disease within Paget’s disease of the sacrum. Further research is needed to assess the true incidence of renal cell carcinoma associated with pagetic bone.

  16. Renal Cell Carcinoma Metastasized to Pagetic Bone.

    PubMed

    Ramirez, Ashley; Liu, Bo; Rop, Baiywo; Edison, Michelle; Valente, Michael; Burt, Jeremy

    2016-01-01

    Paget's disease of the bone, historically known as osteitis deformans, is an uncommon disease typically affecting individuals of European descent. Patients with Paget's disease of the bone are at increased risk for primary bone neoplasms, particularly osteosarcoma. Many cases of metastatic disease to pagetic bone have been reported. However, renal cell carcinoma metastasized to pagetic bone is extremely rare. A 94-year-old male presented to the emergency department complaining of abdominal pain. A computed tomography scan of the abdomen demonstrated a large mass in the right kidney compatible with renal cell carcinoma. The patient was also noted to have Paget's disease of the pelvic bones and sacrum. Within the pagetic bone of the sacrum, there was an enhancing mass compatible with renal cell carcinoma. A subsequent biopsy of the renal lesion confirmed renal cell carcinoma. Paget's disease of the bone places the patient at an increased risk for bone neoplasms. The most commonly reported sites for malignant transformation are the femur, pelvis, and humerus. In cases of malignant transformation, osteosarcoma is the most common diagnosis. Breast, lung, and prostate carcinomas are the most common to metastasize to pagetic bone. Renal cell carcinoma associated with Paget's disease of the bone is very rare, with only one prior reported case. Malignancy in Paget's disease of the bone is uncommon with metastatic disease to pagetic bone being extremely rare. We report a patient diagnosed with concomitant renal cell carcinoma and metastatic disease within Paget's disease of the sacrum. Further research is needed to assess the true incidence of renal cell carcinoma associated with pagetic bone.

  17. Renal Cell Carcinoma Metastasized to Pagetic Bone.

    PubMed

    Ramirez, Ashley; Liu, Bo; Rop, Baiywo; Edison, Michelle; Valente, Michael; Burt, Jeremy

    2016-01-01

    Paget's disease of the bone, historically known as osteitis deformans, is an uncommon disease typically affecting individuals of European descent. Patients with Paget's disease of the bone are at increased risk for primary bone neoplasms, particularly osteosarcoma. Many cases of metastatic disease to pagetic bone have been reported. However, renal cell carcinoma metastasized to pagetic bone is extremely rare. A 94-year-old male presented to the emergency department complaining of abdominal pain. A computed tomography scan of the abdomen demonstrated a large mass in the right kidney compatible with renal cell carcinoma. The patient was also noted to have Paget's disease of the pelvic bones and sacrum. Within the pagetic bone of the sacrum, there was an enhancing mass compatible with renal cell carcinoma. A subsequent biopsy of the renal lesion confirmed renal cell carcinoma. Paget's disease of the bone places the patient at an increased risk for bone neoplasms. The most commonly reported sites for malignant transformation are the femur, pelvis, and humerus. In cases of malignant transformation, osteosarcoma is the most common diagnosis. Breast, lung, and prostate carcinomas are the most common to metastasize to pagetic bone. Renal cell carcinoma associated with Paget's disease of the bone is very rare, with only one prior reported case. Malignancy in Paget's disease of the bone is uncommon with metastatic disease to pagetic bone being extremely rare. We report a patient diagnosed with concomitant renal cell carcinoma and metastatic disease within Paget's disease of the sacrum. Further research is needed to assess the true incidence of renal cell carcinoma associated with pagetic bone. PMID:27660736

  18. Renal Cell Carcinoma Metastasized to Pagetic Bone

    PubMed Central

    Ramirez, Ashley; Liu, Bo; Rop, Baiywo; Edison, Michelle; Valente, Michael

    2016-01-01

    Paget’s disease of the bone, historically known as osteitis deformans, is an uncommon disease typically affecting individuals of European descent. Patients with Paget’s disease of the bone are at increased risk for primary bone neoplasms, particularly osteosarcoma. Many cases of metastatic disease to pagetic bone have been reported. However, renal cell carcinoma metastasized to pagetic bone is extremely rare. A 94-year-old male presented to the emergency department complaining of abdominal pain. A computed tomography scan of the abdomen demonstrated a large mass in the right kidney compatible with renal cell carcinoma. The patient was also noted to have Paget’s disease of the pelvic bones and sacrum. Within the pagetic bone of the sacrum, there was an enhancing mass compatible with renal cell carcinoma. A subsequent biopsy of the renal lesion confirmed renal cell carcinoma. Paget’s disease of the bone places the patient at an increased risk for bone neoplasms. The most commonly reported sites for malignant transformation are the femur, pelvis, and humerus. In cases of malignant transformation, osteosarcoma is the most common diagnosis. Breast, lung, and prostate carcinomas are the most common to metastasize to pagetic bone. Renal cell carcinoma associated with Paget’s disease of the bone is very rare, with only one prior reported case. Malignancy in Paget's disease of the bone is uncommon with metastatic disease to pagetic bone being extremely rare. We report a patient diagnosed with concomitant renal cell carcinoma and metastatic disease within Paget’s disease of the sacrum. Further research is needed to assess the true incidence of renal cell carcinoma associated with pagetic bone. PMID:27660736

  19. Liver Cell Culture Devices

    PubMed Central

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

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    -immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent

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

    PubMed Central

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

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  2. Stem cells in bone tissue engineering.

    PubMed

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

    2010-12-01

    Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have received extensive attention in the field of bone tissue engineering due to their distinct biological capability to differentiate into osteogenic lineages. The application of these stem cells to bone tissue engineering requires inducing in vitro differentiation of these cells into bone forming cells, osteoblasts. For this purpose, efficient in vitro differentiation towards osteogenic lineage requires the development of well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source for application to bone tissue engineering therapies. This review provides a critical examination of the various experimental strategies that could be used to direct the differentiation of ESC, BM-MSC, UCB-MSC, ADSC, MDSC and DPSC towards osteogenic lineages and their potential applications in tissue engineering, particularly in the regeneration of bone.

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

    PubMed Central

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

    2014-01-01

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

  4. Culturing Uveal Melanoma Cells.

    PubMed

    Angi, Martina; Versluis, Mieke; Kalirai, Helen

    2015-04-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-05-01

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

  8. Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defects.

    PubMed

    van der Stok, J; Koolen, M K E; Jahr, H; Kops, N; Waarsing, J H; Weinans, H; van der Jagt, O P

    2014-02-19

    Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.

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

    PubMed Central

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

    2014-01-01

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

  10. Physiological effects of microgravity on bone cells.

    PubMed

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

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    -immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent

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

    PubMed

    Abraham, N G

    1996-12-01

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

  13. Oscillating Cell Culture Bioreactor

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-09-14

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

  18. Double-layered cell transfer technology for bone regeneration

    PubMed Central

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

    2016-01-01

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

  19. Bone Metastasis from Renal Cell Carcinoma.

    PubMed

    Chen, Szu-Chia; Kuo, Po-Lin

    2016-01-01

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

  20. Bone Metastasis from Renal Cell Carcinoma

    PubMed Central

    Chen, Szu-Chia; Kuo, Po-Lin

    2016-01-01

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

  1. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.

    PubMed

    Zhou, Bo O; Yue, Rui; Murphy, Malea M; Peyer, James G; Morrison, Sean J

    2014-08-01

    Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+), 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.

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

    PubMed Central

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

    2013-01-01

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

  3. Characterization of bone marrow derived mesenchymal stem cells in suspension

    PubMed Central

    2012-01-01

    Introduction Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). Methods To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. Conclusions These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs. PMID:23083975

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

    PubMed Central

    Yuan, Jing; Yu, Jian-xiong

    2016-01-01

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

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

    PubMed

    Yuan, Jing; Yu, Jian-Xiong

    2016-05-01

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

  6. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

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

    2014-01-01

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

  7. [Bone and Stem Cells. Immune cell regulation by the bone marrow niche].

    PubMed

    Terashima, Asuka; Takayanagi, Hiroshi

    2014-04-01

    Adult hematopoietic stem cells (HSCs) are maintained in the bone marrow and give rise to all blood cell types. The maintenance and the differentiation of blood cells including immune cells are essential for host defense and oxygen delivery. HSCs are maintained in microenvironments called stem cell niches, which consists of various cell types in bone marrow. Recently, new visualization technologies and assay systems brought advances in studies on the stem cell niche. In addition, several reports demonstrated that osteoblasts and osteocytes regulate not only HSC homeostasis but also immune cell differentiation, suggesting a close relationship between bone cells and HSCs.

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

    PubMed

    de Peppo, Giuseppe Maria; Marolt, Darja

    2014-01-01

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

  9. Impaired function of bone marrow stromal cells in systemic mastocytosis.

    PubMed

    Nemeth, Krisztian; Wilson, Todd M; Ren, Jiaqiang J; Sabatino, Marianna; Stroncek, David M; Krepuska, Miklos; Bai, Yun; Robey, Pamela G; Metcalfe, Dean D; Mezey, Eva

    2015-07-01

    Patients with systemic mastocytosis (SM) have a wide variety of problems, including skeletal abnormalities. The disease results from a mutation of the stem cell receptor (c-kit) in mast cells and we wondered if the function of bone marrow stromal cells (BMSCs; also known as MSCs or mesenchymal stem cells) might be affected by the invasion of bone marrow by mutant mast cells. As expected, BMSCs from SM patients do not have a mutation in c-kit, but they proliferate poorly. In addition, while osteogenic differentiation of the BMSCs seems to be deficient, their adipogenic potential appears to be increased. Since the hematopoietic supportive abilities of BMSCs are also important, we also studied the engraftment in NSG mice of human CD34(+) hematopoietic progenitors, after being co-cultured with BMSCs of healthy volunteers vs. BMSCs derived from patients with SM. BMSCs derived from the bone marrow of patients with SM could not support hematopoiesis to the extent that healthy BMSCs do. Finally, we performed an expression analysis and found significant differences between healthy and SM derived BMSCs in the expression of genes with a variety of functions, including the WNT signaling, ossification, and bone remodeling. We suggest that some of the symptoms associated with SM might be driven by epigenetic changes in BMSCs caused by dysfunctional mast cells in the bone marrow of the patients.

  10. Human progenitor cells for bone engineering applications.

    PubMed

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

    2013-06-01

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

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

    PubMed

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

    2012-06-01

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

  12. Craniofacial defect regeneration using engineered bone marrow mesenchymal stromal cells.

    PubMed

    Yang, Yi; Hallgrimsson, Benedikt; Putnins, Edward E

    2011-10-01

    Large craniofacial bony defects remain a significant clinical challenge. Bone marrow mesenchymal stromal cells (BM-MSCs) constitute a multipotent population. Previously, we developed a novel approach for BM-MSC expansion on 3D CultiSpher-S gelatin microcarrier beads in spin culture with preservation of their multipotentiality, reduction of apoptosis, and enhancement of bone formation in vivo. Here, we hypothesized that such cultured BM-MSCs without exogenous growth factors would respond to the orthopedic microenvironment, thus promoting craniofacial defect regeneration. BM-MSCs isolated from green fluorescent protein (GFP) transgenic rats were ex vivo expanded and transplanted into critical-sized (5-mm diameter) rat calvaria defects. Gelatin beads or defect alone served as controls. By 28 and 42 days, rats were sacrificed for microcomputed tomography (microCT), histologic, and immunohistochemistry examination. MicroCT results demonstrated that BM-MSCs were a statistically significant factor contributing to new bone volume regeneration. Histologic assessment showed that the BM-MSCs group produced more and higher quality new bone compared with beads or defect-alone groups in both osteoinductive and osteoconductive manners. Specifically, immunohistochemical staining identified GFP(+) cells residing in new bone lacunae in conjunction with non-GFP(+) cells. Therefore, ex vivo expanded BM-MSCs at least in part regenerated critical-sized calvaria defects by osteogenic differentiation in vivo.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2015-09-01

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

  15. Reducing bone cancer cell functions using selenium nanocomposites.

    PubMed

    Stolzoff, Michelle; Webster, Thomas J

    2016-02-01

    Cancer recurrence at the site of tumor resection remains a major threat to patient survival despite modern cancer therapeutic advances. Osteosarcoma, in particular, is a very aggressive primary bone cancer that commonly recurs after surgical resection, radiation, and chemotherapeutic treatment. The objective of the present in vitro study was to develop a material that could decrease bone cancer cell recurrence while promoting healthy bone cell functions. Selenium is a natural part of our diet which has shown promise for reducing cancer cell functions, inhibiting bacteria, and promoting healthy cells functions, yet, it has not been widely explored for osteosarcoma applications. For this purpose, due to their increased surface area, selenium nanoparticles (SeNP) were precipitated on a very common orthopedic tissue engineering material, poly-l-lactic acid (or PLLA). Selenium-coated PLLA materials were shown to selectively decrease long-term osteosarcoma cell density while promoting healthy, noncancerous, osteoblast functions (for example, up to two times more alkaline phosphatase activity on selenium coated compared to osteoblasts grown on typical tissue culture plates), suggesting they should be further studied for replacing tumorous bone tissue with healthy bone tissue. Importantly, results of this study were achieved without the use of chemotherapeutics or pharmaceutical agents, which have negative side effects. PMID:26454004

  16. Reducing bone cancer cell functions using selenium nanocomposites.

    PubMed

    Stolzoff, Michelle; Webster, Thomas J

    2016-02-01

    Cancer recurrence at the site of tumor resection remains a major threat to patient survival despite modern cancer therapeutic advances. Osteosarcoma, in particular, is a very aggressive primary bone cancer that commonly recurs after surgical resection, radiation, and chemotherapeutic treatment. The objective of the present in vitro study was to develop a material that could decrease bone cancer cell recurrence while promoting healthy bone cell functions. Selenium is a natural part of our diet which has shown promise for reducing cancer cell functions, inhibiting bacteria, and promoting healthy cells functions, yet, it has not been widely explored for osteosarcoma applications. For this purpose, due to their increased surface area, selenium nanoparticles (SeNP) were precipitated on a very common orthopedic tissue engineering material, poly-l-lactic acid (or PLLA). Selenium-coated PLLA materials were shown to selectively decrease long-term osteosarcoma cell density while promoting healthy, noncancerous, osteoblast functions (for example, up to two times more alkaline phosphatase activity on selenium coated compared to osteoblasts grown on typical tissue culture plates), suggesting they should be further studied for replacing tumorous bone tissue with healthy bone tissue. Importantly, results of this study were achieved without the use of chemotherapeutics or pharmaceutical agents, which have negative side effects.

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

    PubMed

    Glant, T T; Jacobs, J J

    1994-09-01

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

  18. Stimulative effects of lead on bone resorption in organ culture.

    PubMed

    Miyahara, T; Komiyama, H; Miyanishi, A; Takata, M; Nagai, M; Kozuka, H; Hayashi, T; Yamamoto, M; Ito, Y; Odake, H

    1995-03-31

    To clarify whether hypercalcemia after injection of Pb to rats is due to biological bone resorption or physicochemical mineral dissolution, the effect of lead (Pb) on release of previously incorporated 45Ca in organ culture was investigated. Pb at 50 microM and above stimulated the release of 45Ca and hydroxyproline (Hyp). Pb did not stimulate 45Ca release from the bones inactivated by freezing and thawing. Eel calcitonin (ECT), bafilomycin A1 and scopadulcic acid B (SDB) inhibited Pb-stimulated 45Ca release. These results indicate that Pb-induced 45Ca release is due to osteoclastic bone resorption. Pb-stimulated bone resorption was inhibited by indomethacin and flurbiprofen. Pb stimulated the release of prostaglandin E2 (PGE2) from the bones into the media. There was significantly high correlation between 45Ca and PGE2 release. Pb-induced bone resorption was inferred to be mediated by PGE2. From these results, it was suggested that hypercalcemia after Pb injection might be caused by biological bone resorption.

  19. [Cellular interplay of bone cells and vascular endothelial cells in bone].

    PubMed

    Hasegawa, Tomoka; Tsuchiya, Erika; Abe, Miki; Amizuka, Norio

    2016-05-01

    During endochondral bone development, the longitudinal vascular invasion into cartilage primordium initially takes place, by which mineralized cartilage matrix would be exposed into bone. Thereafter, osteogenic cells differentiate into mature osteoblasts to deposit new bone onto the exposed mineralized cartilage. New bone formation at the chondro-osseous junction appears to be achieved by the process of modeling, but not by bone remodeling based on cellular coupling between osteoclasts and osteoblasts. Recently, a specific vessel subtype in bone was reported:Vascular endothelial cells close to the chondro-oseous junction showed intense CD31/Endomucin(CD31(hi)Emcn(hi), type H), while the endothelial cells of sinusoidal vessels in diaphysis revealed only weak CD31/Endomucin(CD31(lo)Emcnlo, type L). It is suggested crucial roles of endothelial HIF in controlling bone angiogenesis, type H vessel abundance, endothelial growth factor expression and osteogenesis.

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

    PubMed

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

    2014-02-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1988-06-01

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

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

    PubMed Central

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

    2013-01-01

    Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×103 cells. To create bone marrow cell-enriched pseudoislets 2×103 islet cells were co-cultured with 2×103 bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation. PMID:23875013

  4. Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Tashjian, A H

    1988-06-01

    Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-05-21

    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.

  7. HOW DO BONE CELLS SENSE MECHANICAL LOADING?

    PubMed Central

    Gusmão, Carlos Vinícius Buarque de; Belangero, William Dias

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1988-11-01

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

  10. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

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

  11. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

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

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

    SciTech Connect

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

    2007-03-09

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

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

    SciTech Connect

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

    1990-02-01

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

  14. Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits.

    PubMed

    Udehiya, Rahul Kumar; Amarpal; Aithal, H P; Kinjavdekar, P; Pawde, A M; Singh, Rajendra; Taru Sharma, G

    2013-06-01

    Autogenic and allogenic bone marrow derived mesenchymal stem cells (BM-MSCs) were compared for repair of bone gap defect in rabbits. BM-MSCs were isolated from bone marrow aspirates and cultured in vitro for allogenic and autogenic transplantation. A 5mm segmental defect was created in mid-diaphysis of the radius bone. The defect was filled with hydroxyapatite alone, hydroxyapatite with autogeneic BM-MSCs and hydroxyapatite with allogenic BM-MSCs in groups A, B and C, respectively. On an average 3.45×10(6) cells were implanted at each defect site. Complete bridging of bone gap with newly formed bone was faster in both treatment groups as compared to control group. Histologically, increased osteogenesis, early and better reorganization of cancellous bone and more bone marrow formation were discernible in treatment groups as compared to control group. It was concluded that in vitro culture expanded allogenic and autogenic BM-MSCs induce similar, but faster and better healing as compared to control.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-04-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. [Bone and Stem Cells. Bone marrow microenvironment niches for hematopoietic stem and progenitor cells].

    PubMed

    Nagasawa, Takashi

    2014-04-01

    In bone marrow, the special microenvironments known as niches control proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) . However, the identity and functions of the niches has been a subject of longstanding debate. Although it has been reported previously that osteoblasts lining the bone surface act as HSC niches, their precise role in HSC maintenance remains unclear. On the other hand, the adipo-osteogenic progenitors with long processes, termed CXCL12-abundant reticular (CAR) cells, which preferentially express the chemokine CXCL12, stem cell factor (SCF) , leptin receptor and PDGF receptor-β were identified in the bone marrow. Recent studies revealed that endothelial cells of bone marrow vascular sinuses and CAR cells provided niches for HSCs. The identity and functions of various other candidate HSC niche cells, including nestin-expressing cells and Schwann cells would also be discussed in this review.

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

    PubMed

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

    2013-04-19

    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

  20. The stem cell niches in bone

    PubMed Central

    Yin, Tong; Li, Linheng

    2006-01-01

    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. PMID:16670760

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Bucur, Florin; Moradi, Babak

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures

    SciTech Connect

    Collins, D.A.; Chambers, T.J. )

    1991-02-01

    Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10{sup {minus} 6}-10{sup {minus} 9} M) and PGE1 (10{sup {minus} 6} - 10{sup {minus} 7} M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Bone resorption was increased (10{sup {minus} 7} M PGE2 and 10{sup {minus} 6} M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Avrunin, A S; Doktorov, A A

    2016-01-01

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

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

    PubMed

    Colnot, Céline

    2009-02-01

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

  9. Recent highlights on bone stem cells: a report from Bone Stem Cells 2009, and not only….

    PubMed

    Cenni, Elisabetta; Perut, Francesca; Baglìo, Serena Rubina; Fiorentini, Elisa; Baldini, Nicola

    2010-11-01

    The use of stem cells has opened new prospects for the treatment of orthopaedic conditions characterized by large bone defects. However, many issues still exist to which answers are needed before routine, large-scale application becomes possible. Bone marrow stromal cells (MSC), which are clonogenic, multipotential precursors present in the bone marrow stroma, are generally employed for bone regeneration. Stem cells with multilineage differentiation similar to MSC have also been demonstrated in adipose tissue, peripheral blood, umbilical cord and amniotic fluid. Each source presents its own advantages and drawbacks. Unfortunately, no unique surface antigen is expressed by MSC, and this hampers simple MSC enrichment from heterogeneous populations. MSC are identified through a combination of physical, morphological and functional assays. Different in vitro and in vivo models have been described for the research on bone stem cells. These models should predict the in vivo bone healing capacity of MSC and if the induced osteogenesis is similar to the physiological one. Although stem cells offer an exciting possibility of a renewable source of cells and tissues for replacement, orthopaedic applications often represent case reports whereas controlled randomized trials are still lacking. Further biological aspects of bone stem cells should be elucidated and a general consensus on the best models, protocols and proper use of scaffolds and growth factors should be achieved.

  10. Ex Vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain.

    PubMed

    David, Valentin; Guignandon, Alain; Martin, Aline; Malaval, Luc; Lafage-Proust, Marie-Hélène; Rattner, Aline; Mann, Val; Noble, Brendon; Jones, David B; Vico, Laurence

    2008-01-01

    Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Young's modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    PubMed Central

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

    2013-01-01

    Introduction Deproteinized bovine bone mineral (Bio-Oss) is a xenogenic bone substitute, widely used in maxillofacial bone regeneration. The aim of this in vitro study was to investigate its influence on the growth behavior of human osteosarcoma cell line, Saos-2 culture, and compare it with the physiologic dose of Dexamethasone, an inductive factor for osteoblasts. Materials and Methods Human osteosarcoma cells, Saos-2, were cultured on Bio-Oss and their growth rate was compared to Saos-2 cultures treated with Dexamethasone 10-7 M in contrast to cells cultivated in PBS, in the control group. Assessment of proliferation was performed after 24, 36, and 48 hours by counting cells using trypan blue exclusion method. Alkaline phosphatase was measured spectrophotometrically at 405 nm with paranitrophenol buffer. Results After 48 hours, the number of Saos-2 cells increased significantly when subcultured with Bio-Oss. Bio-Oss was more effective on the enhancement of proliferation of Saos-2 cells when compared to the physiologic dose of Dexamethasone (P<0.05). Alkaline phosphatase activity increased in cells grown on Bio-Oss and dexamethasone 10-7 M in contrast to cells cultivated in PBS control group. The greatest level of activity was observed in the group containing Bio-Oss after 48 hour. Conclusion The significant increase of cell proliferation and alkaline phosphatase activity in cells cultured on Bio-Oss, compared to Dexamethasone-treated cells, suggests the important role of this bone substitute in promoting bone regeneration. PMID:23922573

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

    SciTech Connect

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

    2012-03-23

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

  14. Ureaplasma infection of cell cultures.

    PubMed Central

    Kotani, H; McGarrity, G J

    1986-01-01

    Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium. Images PMID:3699891

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

    PubMed

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

    2015-01-01

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

  16. Effect of bone marrow-derived stem cells on chondrocytes from patients with osteoarthritis.

    PubMed

    Zhang, Qiangzhi; Chen, Yong; Wang, Qiang; Fang, Chaoyong; Sun, Yu; Yuan, Tao; Wang, Yuebei; Bao, Rongni; Zhao, Ningjian

    2016-02-01

    Increasing numbers of individuals are suffering from osteoarthritis every year, and the directed intra-articular injection of bone marrow stem cells has provided a promising treatment strategy for osteoarthritis. Although a number of studies have demonstrated that intra-articular injection of bone marrow stem cells produced desirable results, the mechanism underlying this effect has not been elucidated. In the current study, the effect of bone marrow stem cells on chondrocytes from patients with osteoarthritis was observed in a co-culture system. Human chondrocytes were obtained from patients with osteoarthritis who underwent surgical procedures and bone marrow stem cells were obtained from bone marrow aspirates, and then the chondrocytes were then cultured alone or cocultured with bone marrow stem cells in 0.4-µm Transwell inserts. The differentiation and biological activity of chondrocytes in the culture system were measured, and the inflammatory factors and OA-associated markers were also measured. The results indicated that coculture with human bone marrow stem cells increases cell proliferation of chondrocytes and inhibits inflammatory activity in osteoarthritis.

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

    PubMed

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

    2011-10-01

    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.

  18. Signaling between tumor cells and the host bone marrow microenvironment.

    PubMed

    Kovacic, Natasa; Croucher, Peter I; McDonald, Michelle M

    2014-01-01

    Tumor cells with high skeletal homing affinity express numerous cell surface receptors that bind ligands produced in bone. Upon arrival, these cells survive in the host environment, encompassed in close proximity to bone marrow cells. Interactions between tumor cells and cells of the host microenvironment are essential to not only tumor cell survival but also their activation and proliferation into environment-modifying tumors. Through the production of RANKL, PTHrP, cytokines, and integrins, activated tumor cells stimulate osteoclastogenesis, enhance bone resorption, and subsequently release matrix-bound proteins that further promote tumor growth and bone resorption. In addition, alterations in the TGF-β/BMP and Wnt signaling pathways via tumor cell growth can either stimulate or suppress osteoblastic bone formation and function, leading to sclerotic or lytic bone disease, respectively. Hence, the presence of tumor cells in bone dysregulates bone remodeling, dramatically impairing skeletal integrity. Furthermore, through complex mechanisms, cells of the immune system interact with tumor cells to further impact bone remodeling. Lastly, with alterations in bone cell activity, the environment is permissive to promoting tumor growth further, suggesting an interdependence between tumor cells and bone cells in metastatic bone disease and multiple myeloma.

  19. Cell Biology of Thiazide Bone Effects

    NASA Astrophysics Data System (ADS)

    Gamba, Gerardo; Riccardi, Daniela

    2008-09-01

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

  20. Comparative characterization of hair follicle dermal stem cells and bone marrow mesenchymal stem cells.

    PubMed

    Hoogduijn, Martin J; Gorjup, Erwin; Genever, Paul G

    2006-02-01

    We compared the growth and differentiation characteristics of hair follicle-derived dermal stem cells with bone marrow mesenchymal stem cells (MSCs). Follicular dermal cells were isolated from whisker hairs of Wistar rats and bone marrow MSCs were isolated from femora of the same animals. The adherent hair follicle dermal cells showed a fibroblastic morphology in serum-containing culture medium, were CD44(+), CD73(+), CD90(+), and CD34(), and had a population doubling time of 27 h. MSCs isolated from the bone marrow showed a similar morphology and population doubling time and expressed the same cell-surface markers. Following exposure to appropriate induction stimuli, both cell populations had the capacity to differentiate into various mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes, and myocytes and expressed neuroprogenitor cell markers. The rate and extent of differentiation were remarkably similar for both hair follicleand bone marrow-derived cells, whereas interfollicular dermal cells failed to differentiate. We identified telomerase activity in follicle dermal stem cells and marrow MSCs and demonstrated that they were capable of clonal expansion. In ex vivo analyses, we identified the presence of putative dermal stem cells in the dermal sheath and dermal papillae of the hair follicle. Consequently, the hair follicle may represent a suitable, accessible source for MSCs.

  1. High density cell culture system

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor)

    1994-01-01

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

  2. Three-dimensional culture may promote cell reprogramming.

    PubMed

    Han, Jin; Chen, Lei; Luo, Guanzheng; Dai, Bin; Wang, Xiujie; Dai, Jianwu

    2013-01-01

    Stem cells reside in stem cells niches, which maintain the balance of self-renewal and differentiation of stem cells. In stem cell niches, cell-cell, cell-extracellular matrix interactions and diffusible signals are important elements. However, another pivotal element is that localized and diffusible signals are all organized as three-dimensional (3-D) structures, which is easily neglected by in vitro cell biology research. Under 3-D culture conditions, the morphology of cells exhibited differently from cultured in traditional two-dimensional (2-D) conditions. Under 3-D culture conditions, the self-renewal and pluripotency of neural stem cells (NSCs) and bone marrow mesenchymal stem cells (BMSCs) were enhanced compared with culturing under 2-D conditions. 3-D cultures could change the transcriptional profile of NSCs compared with 2-D cultures. We hypothesized that 3-D cultures could reprogram mature cells such as fibroblasts to an immature state, like the pluripotent stem cells. The primary results indicated that several ES marker genes were upregulated by 3-D cultures. Though further experiments are needed, this work may provide a method of reprogramming mature cells without gene modifications. PMID:23820263

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

    PubMed

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

    2005-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    SciTech Connect

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

    2013-04-19

    Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2012-12-01

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

  8. Radionuclide bone scanning in giant cell tumor

    SciTech Connect

    Van Nostrand, D.; Madewell, J.E.; McNiesh, L.M.; Kyle, R.W.; Sweet, D.

    1986-03-01

    Radionuclide bone scan findings are described and correlated with pathology in 23 patients with giant cell tumor (GCT) of the bone. The degree of radionuclide activity was markedly increased in 20 (87%), minimally increased in three (13%), and decreased in none of the patients. Of the 23 patients with increased radioactivity, the pattern was diffuse in 11 (48%) and doughnut in 12 (52%). Extended patterns of radioactivity were present in 19 of 22 patients; however, none were associated with true tumor extension. Bone scanning did not aid in the detection of GCT, was nonspecific, and did not differentiate benign from malignant GCT. Although radioactivity extended beyond the radiographic abnormality in the majority of patients, this was most likely secondary to other bony abnormalities or local and/or regional hyperemia, and caution should be taken in ascribing this extension to either tumor or metastasis.

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  10. Stromal cells and stem cells in clinical bone regeneration

    PubMed Central

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

    2015-01-01

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

  11. Suspension culture of mammalian cells.

    PubMed

    Birch, J R; Arathoon, R

    1990-01-01

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

  12. Bone formation in vitro and in nude mice by human osteosarcoma cells.

    PubMed

    Ogose, A; Motoyama, T; Hotta, T; Watanabe, H; Takahashi, H E

    1995-01-01

    Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Lawless, Desales

    1989-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed Central

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

    2014-01-01

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

  19. Cell culture purity issues and DFAT cells

    SciTech Connect

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

    2013-04-12

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

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

    PubMed

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

    1990-10-01

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

  1. Aged human bone marrow stromal cells maintaining bone forming capacity in vivo evaluated using an improved method of visualization.

    PubMed

    Stenderup, K; Rosada, C; Justesen, J; Al-Soubky, T; Dagnaes-Hansen, F; Kassem, M

    2004-01-01

    Age-related decreased osteoblast function is a well-known but poorly understood phenomenon. Previous studies that examined the effects of donor age on osteoblast functions employed in vitro assays that may not reflect the true osteoblast capacity for bone formation. Thus, we have developed an in vivo assay for quantifying the bone forming capacity (BFC) and we compared the BFC of osteoblastic cells obtained from young and old donors. Osteoblasts were obtained from human bone marrow stromal cell cultures and implanted subcutaneously in immuno-deficient mice (NOD/LtSz- Prkdc(scid)). After 8 weeks, the implants were removed and embedded un-decalcified in methyl methacrylate (MMA). Sections were stained histochemically with Goldner's Trichrome stain and immuno-histochemically using human-specific antibodies against known osteogenic markers. Implanted human marrow stromal cells (hMSC) were able to form bone in vivo. The donor origin of bone was verified using several human-specific antibodies. Dose-response experiments demonstrated that 5 x 10(5) hMSC per implant gave the maximal bone formation after 8 weeks. No difference in BFC was observed between cells obtained from young (24-30 years old; mean age 27 +/- 2 years, n = 5) and old (71-81 years old; mean age 75 +/- 4 years, n = 5) donors. Our study demonstrates that the capacity of hMSC to form bone in vivo is maintained with age and suggests that the observed senescence-associated decrease in bone formation is due to a defect in the bone microenvironment, the nature of which remains to be determined.

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

    SciTech Connect

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

    2007-12-31

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

  3. Stem Cells in Teeth and Craniofacial Bones

    PubMed Central

    Zhao, H.; Chai, Y.

    2015-01-01

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

  4. Giant Cell Tumor of Bone - An Overview

    PubMed Central

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

    2016-01-01

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

  5. [Pulmonary arterial hypertension, bone marrow, endothelial cell precursors and serotonin].

    PubMed

    Ayme-Dietrich, Estelle; Banas, Sophie M; Monassier, Laurent; Maroteaux, Luc

    2016-01-01

    Serotonin and bone-marrow-derived stem cells participate together in triggering pulmonary hypertension. Our work has shown that the absence of 5-HT2B receptors generates permanent changes in the composition of the blood and bone-marrow in the myeloid lineages, particularly in endothelial cell progenitors. The initial functions of 5-HT2B receptors in pulmonary arterial hypertension (PAH) are restricted to bone-marrow cells. They contribute to the differentiation/proliferation/mobilization of endothelial progenitor cells from the bone-marrow. Those bone-marrow-derived cells have a critical role in the development of pulmonary hypertension and pulmonary vascular remodeling. These data indicate that bone-marrow derived endothelial progenitors play a key role in the pathogenesis of PAH and suggest that interactions involving serotonin and bone morphogenic protein type 2 receptor (BMPR2) could take place at the level of the bone-marrow. PMID:27687599

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-26

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

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

    PubMed

    Avrunin, A S; Doktorov, A A

    2016-01-01

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

  9. Nanostructured magnesium increases bone cell density

    NASA Astrophysics Data System (ADS)

    Weng, Lucy; Webster, Thomas J.

    2012-12-01

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

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

    PubMed

    Ong, S M; Taylor, G J S

    2003-04-01

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

  11. Heparin modulates intracellular cyclic AMP in human trabecular bone cells and adherent rheumatoid synovial cells.

    PubMed Central

    Crisp, A J; Roelke, M S; Goldring, S R; Krane, S M

    1984-01-01

    Cells were cultured from explants of human trabecular bone excised from eight patients and incubated usually for 20 minutes with bovine parathyroid hormone, salmon calcitonin, prostaglandin E2, or heparin. The intracellular content of cyclic AMP was measured by radioimmunoassay and was significantly increased by parathyroid hormone in four, by calcitonin in two, by prostaglandin E2 in eight, and by heparin in seven out of eight cultures. In the two cultures containing calcitonin-responsive cells heparin inhibited the cyclic AMP response induced by calcitonin. Heparin did not affect the cyclic AMP response to parathyroid hormone or prostaglandin E2. Heparin also increased the cyclic AMP content of cultured adherent rheumatoid synovial cells. It is proposed that, in certain situations of focal pathological bone resorption, although concentrations of circulating hormones may be normal, the local release of products such as heparin may modify the effect of hormones which regulate connective tissue homoeostasis. local changes in hormone responses could contribute to the enhanced bone resorption associated with inflammatory processes such as rheumatoid arthritis. Images PMID:6089675

  12. Bone homing of mesenchymal stem cells by ectopic alpha 4 integrin expression.

    PubMed

    Kumar, Sanjay; Ponnazhagan, Selvarangan

    2007-12-01

    The pluripotent nature of mesenchymal stem cells (MSC) widens their potential for tissue regeneration and as vehicles for cell therapy in molecular medicine. Although the MSC are relatively easier to obtain and propagate in culture, a major impediment remains in their engraftment to target tissues on autologous transfer. We report here that transient, ectopic expression of alpha4 integrin (CD49d) on MSC greatly increases bone homing in an immunocompetent mouse model. Heterodimerization of the alpha4 integrin with endogenous beta1 integrin (CD29) was confirmed to influence this targeting. In addition to retaining their stem cell property, the engrafted MSC were also found to form osteoblasts and osteocytes in the growth plate of recipient mouse limb bones (femur/tibia) in vivo. These findings provide evidence for a novel strategy to achieve bone homing of genetically engineered MSC, which may broadly benefit in targeted therapies for osteopenic bone defects and cancer bone metastasis.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-01-01

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

  15. Connecting mechanics and bone cell activities in the bone remodeling process: an integrated finite element modeling.

    PubMed

    Hambli, Ridha

    2014-01-01

    Bone adaptation occurs as a response to external loadings and involves bone resorption by osteoclasts followed by the formation of new bone by osteoblasts. It is directly triggered by the transduction phase by osteocytes embedded within the bone matrix. The bone remodeling process is governed by the interactions between osteoblasts and osteoclasts through the expression of several autocrine and paracrine factors that control bone cell populations and their relative rate of differentiation and proliferation. A review of the literature shows that despite the progress in bone remodeling simulation using the finite element (FE) method, there is still a lack of predictive models that explicitly consider the interaction between osteoblasts and osteoclasts combined with the mechanical response of bone. The current study attempts to develop an FE model to describe the bone remodeling process, taking into consideration the activities of osteoclasts and osteoblasts. The mechanical behavior of bone is described by taking into account the bone material fatigue damage accumulation and mineralization. A coupled strain-damage stimulus function is proposed, which controls the level of autocrine and paracrine factors. The cellular behavior is based on Komarova et al.'s (2003) dynamic law, which describes the autocrine and paracrine interactions between osteoblasts and osteoclasts and computes cell population dynamics and changes in bone mass at a discrete site of bone remodeling. Therefore, when an external mechanical stress is applied, bone formation and resorption is governed by cells dynamic rather than adaptive elasticity approaches. The proposed FE model has been implemented in the FE code Abaqus (UMAT routine). An example of human proximal femur is investigated using the model developed. The model was able to predict final human proximal femur adaptation similar to the patterns observed in a human proximal femur. The results obtained reveal complex spatio-temporal bone

  16. Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum.

    PubMed

    Nakahara, H; Bruder, S P; Haynesworth, S E; Holecek, J J; Baber, M A; Goldberg, V M; Caplan, A I

    1990-01-01

    Periosteal cells were enzymatically isolated from the tibiae of young chicks, introduced into cell culture, allowed to reach confluence, and subcultured. The freshly isolated or subcultured cells were loaded into diffusion chambers and implanted into the peritoneal cavity of athymic mice to test their osteo-chondrogenic potential in a contained in vivo location. Freshly isolated periosteal cells formed both bone and cartilage tissue in such test chambers, but with a relatively low incidence. In contrast, cultured periosteal cells consistently gave rise to bone and cartilage even after 10 population doublings. With further passages of cells, the osteo-chondrogenic potential diminished substantially, until complete loss of expressivity at 16 population doublings or longer. Cultured muscle fibroblasts, when loaded into diffusion chambers under identical conditions to those of cultured periosteal cells, formed neither bone nor cartilage. These observations suggest that periosteal cells of young chicks contain subsets of progenitor cells or mesenchymal stem cells which possess the potential to differentiate into osteoblasts or chondrocytes, and this potential is retained after enzymatic isolation and for several population doublings in culture.

  17. Bone marrow transplantation in sickle cell anaemia.

    PubMed Central

    Vermylen, C; Cornu, G; Philippe, M; Ninane, J; Borja, A; Latinne, D; Ferrant, A; Michaux, J L; Sokal, G

    1991-01-01

    Sickle cell anaemia is still responsible for severe crippling and death in young patients living in developing countries. Apart from prophylaxis and treatment of infections, no active treatment can be safely proposed in such areas of the world. Therefore a bone marrow transplantation was performed in 12 patients staying in Belgium and planning to return to Africa. Twelve patients, aged between 11 months and 23 years (median 4 years), underwent a HLA identical bone marrow transplantation. The conditioning regimen included oral busulphan for four consecutive days (4 mg/kg) followed by four days of intravenous cyclophosphamide (50 mg/kg). In 10 patients the engraftment was rapid and sustained. A further patient suffered transient red cell hypoplasia and another underwent a second bone marrow transplantation from the same donor at day 62 because of graft rejection. All patients are alive and well with a follow up ranging from 9-51 months (median 27 months). In all cases a complete cessation of vaso-occlusive episodes and haemolysis was observed as was a change in the haemoglobin pattern in accordance with the donor's electrophoretic pattern. PMID:1953001

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

    PubMed Central

    Bodle, Josephine C.; Hanson, Ariel D.

    2011-01-01

    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. PMID:21338267

  19. Imaging Sensitivity of Quiescent Cancer Cells to Metabolic Perturbations in Bone Marrow Spheroids

    PubMed Central

    Cavnar, Stephen P.; Xiao, Annie; Gibbons, Anne E.; Rickelmann, Andrew D.; Neely, Taylor; Luker, Kathryn E.; Takayama, Shuichi; Luker, Gary D.

    2016-01-01

    Malignant cells from breast cancer and other common cancers such as prostate and melanoma may persist in bone marrow as quiescent, non-dividing cells that remain viable for years or even decades before resuming proliferation to cause recurrent disease. This phenomenon, referred to clinically as tumor dormancy, poses tremendous challenges to curing patients with breast cancer. Quiescent tumor cells resist chemotherapy drugs that predominantly target proliferating cells, limiting success of neo-adjuvant and adjuvant therapies. We recently developed a 3D spheroid model of quiescent breast cancer cells in bone marrow for mechanistic and drug testing studies. We combined this model with optical imaging methods for label-free detection of cells preferentially utilizing glycolysis versus oxidative metabolism to investigate the metabolic state of co-culture spheroids with different bone marrow stromal and breast cancer cells. Through imaging and biochemical assays, we identified different metabolic states of bone marrow stromal cells that control metabolic status and flexibilities of co-cultured breast cancer cells. We tested metabolic stresses and targeted inhibition of specific metabolic pathways to identify approaches to preferentially eliminate quiescent breast cancer cells from bone marrow environments. These studies establish an integrated imaging approach to analyze metabolism in complex tissue environments to identify new metabolically-targeted cancer therapies. PMID:27478871

  20. Short-time survival and engraftment of bone marrow stromal cells in an ectopic model of bone regeneration.

    PubMed

    Giannoni, Paolo; Scaglione, Silvia; Daga, Antonio; Ilengo, Cristina; Cilli, Michele; Quarto, Rodolfo

    2010-02-01

    In tissue-engineered applications bone marrow stromal cells (BMSCs) are combined with scaffolds to target bone regeneration; animal models have been devised and the cells' long-term engraftment has been widely studied. However, in regenerated bone, the cell number is severely reduced with respect to the initially seeded BMSCs. This reflects the natural low cellularity of bone but underlines the selectivity of the differentiation processes. In this respect, we evaluated the short-term survival of BMSCs, transduced with the luciferase gene, after implantation of cell-seeded scaffolds in a nude mouse model. Cell proliferation/survival was assessed by bioluminescence imaging: light production was decreased by 30% on the first day, reaching a 50% loss within 48 h. Less than 5% of the initial signal remained after 2 months in vivo. Apoptotic BMSCs were detected within the first 2 days of implantation. Interestingly, the initial frequency of clonogenic progenitors matched the percentage of in vivo surviving cells. Cytokines and inflammation may contribute to the apoptotic onset at the implant milieu. However, preculturing cells with tumor necrosis factor alpha enhanced survival, allowing detection of 8.1% of the seeded BMSCs 2 months after implantation. Thus culturing conditions may reduce the apoptotic overload of seeded osteoprogenitors, strengthening the constructs' osteogenic potential.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. The survival of cryopreserved human bone marrow stem cells.

    PubMed

    Hill, R S; Mackinder, C A; Postlewaight, B F; Blacklock, H A

    1979-07-01

    Two methods for cryopreservation of bone marrow stem cells were compared using bone marrow obtained from 36 patients. Included in this group were 21 persons with the diagnosis of leukaemia including 14 either with acute myeloid or lymphoblastic leukaemia in remission following intensive remission induction chemotherapy. After freeze-preservation and reconstitution, all marrow samples were tested for nucleated cell (NC) recovery and grown on agar to assess colony forming units (CFUC) and cluster forming units in culture (CluFUc). A slow dilution reconstitution method using freezing media containing AB negative plasma resulted in recovery of 85% of the CFUc activity of fresh marrow. This result was significantly better than the 47% CFUc recovery obtained when freezing media without plasma and a rapid dilution reconstitution technique were used. NC recoveries following slow dilution (51%) and rapid dilution (44%) were not significantly different. CluFUc were disproportionately reduced compared with CFUc although yielding similar results with both methods (26% and 32%). No correlation was found for either method between CFUc and NC recovery or between CFUc and CluFUc recovery in cryopreserved bone marrow. PMID:392422

  4. Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells.

    PubMed

    Koerner, Jens; Nesic, Dobrila; Romero, Jose Diaz; Brehm, Walter; Mainil-Varlet, Pierre; Grogan, Shawn Patrick

    2006-06-01

    Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

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

    PubMed

    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

    2004-05-01

    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.

  6. Spine Fusion Using Cell Matrix Composites Enriched in Bone Marrow-Derived Cells

    PubMed Central

    Nitto, Hironori; Matsukura, Yoichi; Boehm, Cynthia; Valdevit, Antonio; Kambic, Helen; Davros, William; Powell, Kimerly; Easley, Kirk

    2005-01-01

    Bone marrow-derived cells including osteoblastic progenitors can be concentrated rapidly from bone marrow aspirates using the surface of selected implantable matrices for selective cell attachment. Concentration of cells in this way to produce an enriched cellular composite graft improves graft efficacy. The current study was designed to test the hypothesis that the biologic milieu of a bone marrow clot will significantly improve the efficacy of such a graft. An established posterior spinal fusion model and cancellous bone matrix was used to compare an enriched cellular composite bone graft alone, bone matrix plus bone marrow clot, and an enriched bone matrix composite graft plus bone marrow clot. Union score, quantitative computed tomography, and mechanical testing were used to define outcome. The union score for the enriched bone matrix plus bone marrow clot composite was superior to the enriched bone matrix alone and the bone matrix plus bone marrow clot. The enriched bone matrix plus bone marrow clot composite also was superior to the enriched bone matrix alone in fusion volume and in fusion area. These data confirm that the addition of a bone marrow clot to an enriched cell-matrix composite graft results in significant improvement in graft performance. Enriched composite grafts prepared using this strategy provide a rapid, simple, safe, and inexpensive method for intraoperative concentration and delivery of bone marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting. PMID:12567137

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

    PubMed

    Bouet, G; Cruel, M; Laurent, C; Vico, L; Malaval, L; Marchat, D

    2015-01-01

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

  8. Cell culture compositions

    DOEpatents

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

    2014-03-18

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

  9. Catch-up growth after dexamethasone withdrawal occurs in cultured postnatal rat metatarsal bones.

    PubMed

    Chagin, Andrei S; Karimian, Elham; Sundström, Katja; Eriksson, Emma; Sävendahl, Lars

    2010-01-01

    Children exposed to systemic glucocorticoids often exhibit growth retardation and after the cessation of therapy catch-up growth occurs in many, but not all patients. The developmental regulation and underlying cellular mechanisms of catch-up growth are not fully understood. To clarify this issue, we established an in vitro model of catch-up growth. Here we present a protocol for the long-term culture (up to 160 days) of fetal (E20) as well as postnatal (P8) rat metatarsal bones which allowed us to characterize ex vivo the phenomenon of catch-up growth without any influence by systemic factors. The relevance of the model was confirmed by the demonstration that the growth of fetal and postnatal bones were stimulated by IGF1 (100 ng/ml) and inhibited by dexamethasone (Dexa; 1 microM). We found that the capacity to undergo catch-up growth was restricted to postnatal bones. Catch-up growth occurred after postnatal bones had been exposed to Dexa for 7 or 12 days but not after a more prolonged exposure (19 days). Incomplete catch-up growth resulted in compromised bone length when assessed at the end of the 4-month period of culture. While exposure to Dexa was associated with decreased chondrocyte proliferation and differentiation, catch-up growth was only associated with increased cell proliferation. We conclude that the phenomenon of catch-up growth after Dexa treatment is intrinsic to the growth plate and primarily mediated by an upregulation of chondrocyte proliferation.

  10. Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

    PubMed

    Mathews, Smitha; Mathew, Suja Ann; Gupta, Pawan Kumar; Bhonde, Ramesh; Totey, Satish

    2014-02-01

    Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell-based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan-treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin-4-sulphate. Osteoblast differentiation-associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin-6-sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering.

  11. Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during the bone repair process.

    PubMed

    Okada, Kiyotaka; Kawao, Naoyuki; Yano, Masato; Tamura, Yukinori; Kurashimo, Shinzi; Okumoto, Katsumi; Kojima, Kotarou; Kaji, Hiroshi

    2016-01-01

    Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

  12. Differentiation of macrophages from normal human bone marrow in liquid culture. Electron microscopy and cytochemistry.

    PubMed Central

    Bainton, D R; Golde, D W

    1978-01-01

    To study the various stages of human mononuclear phagocyte maturation, we cultivated bone marrow in an in vitro diffusion chamber with the cells growing in suspension and upon a dialysis membrane. At 2, 7, and 14 days, the cultured cells were examined by electron microscopy and cytochemical techniques for peroxidase and for more limited analysis of acid phosphatase and arylsulfatase. Peroxidase was being synthesized in promonocytes of 2- and 7-day cultures, as evidenced by reaction product in the rough-surfaced endoplasmic reticulum, Golgi complex, and storage granules. Peroxidase synthesis had ceased in monocytes and the enzyme appeared only in some granules. By 7 days, large macrophages predominated, containing numerous peroxidase-positive storage granules, and heterophagy of dying cells was evident. By 14 days, the most prevalent cell type was the large peroxidase-negative macrophage. Thus, peroxidase is present in high concentrations in immature cells but absent at later stages, presumably a result of degranulation of peroxidase-positive storage granules. Clusters of peroxidase-negative macrophages with indistinct borders (epithelioid cells), as well as obvious multinucleated giant cells, were noted. Frequently, the interdigitating plasma membranes of neighboring macrophages showed a modification resembling a septate junction--to our knowledge, representing the first documentation of this specialized cell contact between normal macrophages. We suggest that such junctions may serve as zones of adhesion between epithelioid cells. Images PMID:659615

  13. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.

    PubMed

    Yu, Vionnie W C; Saez, Borja; Cook, Colleen; Lotinun, Sutada; Pardo-Saganta, Ana; Wang, Ying-Hua; Lymperi, Stefania; Ferraro, Francesca; Raaijmakers, Marc H G P; Wu, Joy Y; Zhou, Lan; Rajagopal, Jayaraj; Kronenberg, Henry M; Baron, Roland; Scadden, David T

    2015-05-01

    Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn(+) cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell-based adaptive immunity.

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

    PubMed Central

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

    2016-01-01

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

  15. Cartilage Repair With Autologous Bone Marrow Mesenchymal Stem Cell Transplantation

    PubMed Central

    Yamasaki, Shinya; Mera, Hisashi; Itokazu, Maki; Hashimoto, Yusuke

    2014-01-01

    Clinical trials of various procedures, including bone marrow stimulation, mosaicplasty, and autologous chondrocyte implantation, have been explored to treat articular cartilage defects. However, all of them have some demerits. We focused on autologous culture-expanded bone marrow mesenchymal stem cells (BMSC), which can proliferate without losing their capacity for differentiation. First, we transplanted BMSC into the defective articular cartilage of rabbit and succeeded in regenerating osteochondral tissue. We then applied this transplantation in humans. Our previous reports showed that treatment with BMSC relieves the clinical symptoms of chondral defects in the knee and elbow joint. We investigated the efficacy of BMSC for osteoarthritic knee treated with high tibial osteotomy, by comparing 12 BMSC-transplanted patients with 12 cell-free patients. At 16-month follow-up, although the difference in clinical improvement between both groups was not significant, the arthroscopic and histological grading score was better in the cell-transplanted group. At the over 10-year follow-up, Hospital for Special Surgery knee scores improved to 76 and 73 in the BMSC-transplanted and cell-free groups, respectively, which were better than preoperative scores. Additionally, neither tumors nor infections were observed in all patients, and in the clinical study, we have never observed hypertrophy of repaired tissue, thereby guaranteeing the clinical safety of this therapy. Although we have never observed calcification above the tidemark in rabbit model and human histologically, the repair cartilage was not completely hyaline cartilage. To elucidate the optimum conditions for cell therapy, other stem cells, culture conditions, growth factors, and gene transfection methods should be explored. PMID:26069698

  16. CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

    PubMed Central

    Yang, Junjie; Ii, Masaaki; Kamei, Naosuke; Alev, Cantas; Kwon, Sang-Mo; Kawamoto, Atsuhiko; Akimaru, Hiroshi; Masuda, Haruchika; Sawa, Yoshiki; Asahara, Takayuki

    2011-01-01

    Background Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. Methodology/Principal Findings CD34+ cells, c-Kit+/Sca-1+/Lin− (KSL) cells, c-Kit+/Lin− (KL) cells and Sca-1+/Lin− (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34+ cells showed the lowest EPC colony forming activity, CD34+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. Conclusion These findings suggest that mouse CD34+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology. PMID:21655289

  17. Synergistic effect of scaffold composition and dynamic culturing environment in multilayered systems for bone tissue engineering.

    PubMed

    Rodrigues, Márcia T; Martins, Albino; Dias, Isabel R; Viegas, Carlos A; Neves, Nuno M; Gomes, Manuela E; Reis, Rui L

    2012-11-01

    Bone extracellular matrix (ECM) is composed of mineralized collagen fibrils which support biological apatite nucleation that participates in bone outstanding properties. Understanding and mimicking bone morphological and physiological parameters at a biological scale is a major challenge in tissue engineering scaffolding. Using emergent (nano)technologies scaffold designing may be critically improved, enabling highly functional tissue substitutes for bone applications. This study aims to develop novel biodegradable composite scaffolds of tricalcium phosphate (TCPs) and electrospun nanofibers of poly(ϵ-caprolactone) (PCL), combining TCPs osteoconductivity with PCL biocompatibility and elasticity, mimicking bone structure and composition. We hypothesized that scaffolds with such structure/composition would stimulate the proliferation and differentiation of bone marrow stromal cells (BMSCs) towards the osteogenic phenotype. Composite scaffolds, developed by electrospining using consecutive stacked layers of PCL and TCPs, were characterized by FTIR spectroscopy, X-Ray diffraction and scanning electronic microscopy. Cellular behavior was assessed in goat BMSCs seeded onto composite scaffolds and cultured in static or dynamic conditions, using basal or osteogenic media during 7, 14 or 21 days. Cellular proliferation was quantified and osteogenic differentiation confirmed by alkaline phosphatase activity, alizarin red staining and immunocytochemistry for osteocalcin and collagen I. Results suggest that PCL-TCP scaffolds provide a 3D support for gBMSCs proliferation and osteogenic differentiation with production of ECM. TCPs positively stimulate the osteogenic process, especially under dynamic conditions, where PCL-TCP scaffolds are sufficient to promote osteogenic differentiation even in basal medium conditions. The enhancement of the osteogenic potential in dynamic conditions evidences the synergistic effect of scaffold composition and dynamic stimulation in g

  18. Identification of Rorβ targets in cultured osteoblasts and in human bone

    SciTech Connect

    Roforth, Matthew M. Khosla, Sundeep Monroe, David G.

    2013-11-01

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

  19. Cytokines and growth factors which regulate bone cell function

    NASA Astrophysics Data System (ADS)

    Seino, Yoshiki

    Everybody knows that growth factors are most important in making bone. Hormones enhance bone formation from a long distance. Growth factors promote bone formation as an autocrine or paracrine factor in nearby bone. BMP-2 through BMP-8 are in the TGF-β family. BMP makes bone by enchondral ossification. In bone, IGF-II is most abundant, second, TGF-β, and third IGF-I. TGF-β enhances bone formation mainly by intramembranous ossification in vivo. TGF-β affects both cell proliferation and differentiation, however, TGF-β mainly enhances bone formation by intramembranous ossification. Interestingly, TGF-β is increased by estrogen(E 2), androgen, vitamin D, TGF-β and FGF. IGF-I and IGF-II also enhance bone formation. At present it remains unclear why IGF-I is more active in bone formation than IGF-II, although IGF-II is more abundant in bone compared to IGF-I. However, if only type I receptor signal transduction promotes bone formation, the strong activity of IGF-I in bone formation is understandable. GH, PTH and E 2 promotes IGF-I production. Recent data suggest that hormones containing vitamin D or E 2 enhance bone formation through growth factors. Therefore, growth factors are the key to clarifying the mechanism of bone formation.

  20. Cell Fate and Differentiation of Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Jimi, Eijiro

    2016-01-01

    Osteoblasts and bone marrow adipocytes originate from bone marrow mesenchymal stem cells (BMMSCs) and there appears to be a reciprocal relationship between adipogenesis and osteoblastogenesis. Alterations in the balance between adipogenesis and osteoblastogenesis in BMMSCs wherein adipogenesis is increased relative to osteoblastogenesis are associated with decreased bone quality and quantity. Several proteins have been reported to regulate this reciprocal relationship but the exact nature of the signals regulating the balance between osteoblast and adipocyte formation within the bone marrow space remains to be determined. In this review, we focus on the role of Transducin-Like Enhancer of Split 3 (TLE3), which was recently reported to regulate the balance between osteoblast and adipocyte formation from BMMSCs. We also discuss evidence implicating canonical Wnt signalling, which plays important roles in both adipogenesis and osteoblastogenesis, in regulating TLE3 expression. Currently, there is demand for new effective therapies that target the stimulation of osteoblast differentiation to enhance bone formation. We speculate that reducing TLE3 expression or activity in BMMSCs could be a useful approach towards increasing osteoblast numbers and reducing adipogenesis in the bone marrow environment. PMID:27298623

  1. Injectable bone tissue engineering using expanded mesenchymal stem cells.

    PubMed

    Yamada, Yoichi; Nakamura, Sayaka; Ito, Kenji; Umemura, Eri; Hara, Kenji; Nagasaka, Tetsuro; Abe, Akihiro; Baba, Shunsuke; Furuichi, Yasushi; Izumi, Yuichi; Klein, Ophir D; Wakabayashi, Toshihiko

    2013-03-01

    Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue-engineered bone (TEB) composed of mesenchymal stem cells (MSCs) and platelet-rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out large-scale clinical studies in patients with long-term follow-up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months were significantly increased over the preoperation baseline (p < .001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long-term follow-up. Injectable TEB restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering.

  2. Mimicking the nanofeatures of bone increases bone-forming cell adhesion and proliferation

    NASA Astrophysics Data System (ADS)

    Palin, Erica; Liu, Huinan; Webster, Thomas J.

    2005-09-01

    There is a great need to design better orthopaedic implant devices by modifying their surface properties. In this respect, one approach that has received much attention of late is the simulation of the surface roughness of bone in synthetic orthopaedic implant materials. Bone has numerous nanometre features due to the presence of nanostructured entities such as collagen and hydroxyapatite. Despite this fact, current orthopaedic implant materials are smooth at the nanoscale. Previous studies have measured increased osteoblast (bone-forming cell) functions on biologically inspired nanophase titania compared to conventional titania formulations. In fact, in vitro calcium deposition by osteoblasts was up to three times higher on nanostructured compared to conventional titania. However, it was unclear in those studies what underlying surface properties (roughness, crystallinity, crystal phase, chemistry, etc) promoted enhanced functions of osteoblasts on nanophase titania. For that reason, the objective of the present in vitro study was to specifically determine the role nanostructured surface roughness of titania had on increasing functions of osteoblasts. To achieve this, the surface roughness of nanophase and conventional titania was transferred to a model tissue engineering polymer: poly-lactic-co-glycolic acid (PLGA). Results of the present study demonstrated greater osteoblast adhesion and proliferation for up to 5 days of culture on PLGA moulds of nanophase compared to conventional titania. In this manner, this study elucidated that the property of nanophase titania which increased osteoblast function was a large degree of nanometre surface features that mimicked bone. For this reason, nanophase materials deserve more attention in improving orthopaedic implant applications.

  3. Proliferative activity of vervet monkey bone marrow-derived adherent cells

    SciTech Connect

    Kramvis, A.; Garnett, H.M.

    1987-11-01

    Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and n = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus, the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

  4. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow

    PubMed Central

    Yu, Vionnie W.C.; Saez, Borja; Cook, Colleen; Lotinun, Sutada; Pardo-Saganta, Ana; Wang, Ying-Hua; Lymperi, Stefania; Ferraro, Francesca; Raaijmakers, Marc H.G.P.; Wu, Joy Y.; Zhou, Lan; Rajagopal, Jayaraj; Kronenberg, Henry M.; Baron, Roland

    2015-01-01

    Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn+ cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell–based adaptive immunity. PMID:25918341

  5. Langerhans cell histiocytosis of bone: MR imaging.

    PubMed

    George, J C; Buckwalter, K A; Cohen, M D; Edwards, M K; Smith, R R

    1994-01-01

    Magnetic resonance (MR) images of 12 pathologically proven lesions of Langerhans cell histiocytosis (LCH) of bone were reviewed retrospectively. MR identified all lesions, three of which were not identified on plain radiographs. In all cases, MR showed greater abnormality than did plain radiographs. With one exception, all lesions were hypointense on T1-weighted images and hyperintense on T2-weighted images. The lesions and associated soft tissue abnormalities were very conspicuous on short TI inversion sequences and T1-weighted post-contrast images. Follow-up MR studies in two patients after chemotherapy showed decreased size and enhancement of lesions compared with baseline studies.

  6. Identification of molecular markers related to human alveolar bone cells and pathway analysis in diabetic patients.

    PubMed

    Sun, X; Ren, Q H; Bai, L; Feng, Q

    2015-10-28

    Alveolar bone osteoblasts are widely used in dental and related research. They are easily affected by systemic diseases such as diabetes. However, the mechanism of diabetes-induced alveolar bone absorption remains unclear. This study systematically explored the changes in human alveolar bone cell-related gene expression and biological pathways, which may facilitate the investigation of its mechanism. Alveolar bone osteoblasts isolated from 5 male diabetics and 5 male healthy adults were cultured. Total RNA was extracted from these cells and subjected to gene microarray analysis. Differentially expressed genes were screened, and a gene interaction network was constructed. An enrichment pathway analysis was simultaneously performed on differentially expressed genes to identify the biological pathways associated with changes in the alveolar bone cells of diabetic humans. In total, we identified 147 mRNAs that were differentially expressed in diabetic alveolar bone cells (than in the normal cells; 91 upregulated and 36 downregulated mRNAs). The constructed co-expression network showed 3 pairs of significantly-expressed genes. High-enrichment pathway analysis identified 8 pathways that were affected by changes in gene expression; three of the significant pathways were related to metabolism (inositol phosphate metabolism, propanoate metabolism, and pyruvate metabolism). Here, we identified a few potential genes and biological pathways for the diagnosis and treatment of alveolar bone cells in diabetic patients.

  7. Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche1

    PubMed Central

    Templeton, Zach S.; Lie, Wen-Rong; Wang, Weiqi; Rosenberg-Hasson, Yael; Alluri, Rajiv V.; Tamaresis, John S.; Bachmann, Michael H.; Lee, Kitty; Maloney, William J.; Contag, Christopher H.; King, Bonnie L.

    2015-01-01

    BACKGROUND/OBJECTIVES: Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche. METHODS: Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry. RESULTS: Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1β (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment. CONCLUSIONS: Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1β, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche. PMID:26696367

  8. Immunomodulation regulates mesenchymal stem cell-based bone regeneration.

    PubMed

    Su, Y; Shi, S; Liu, Y

    2014-10-01

    Mesenchymal stem cell (MSC)-based regenerative medicine represents a promising frontier for bone reconstruction. Significant efforts have been devoted to clarifying the capacities of MSCs to repair or reconstruct bone tissue. This review provides a concise summary of current knowledge pertaining to the possible mechanisms of MSC action in the regeneration of bone, with particular focus on the interplay between donor MSCs and host immune response in the process of new bone regeneration.

  9. 3D Porous Calcium-Alginate Scaffolds Cell Culture System Improved Human Osteoblast Cell Clusters for Cell Therapy

    PubMed Central

    Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

    2015-01-01

    Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects. PMID:25825603

  10. Cementum matrix formation in vivo by cultured dental follicle cells.

    PubMed

    Handa, K; Saito, M; Yamauchi, M; Kiyono, T; Sato, S; Teranaka, T; Sampath Narayanan, A

    2002-11-01

    Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis. PMID:12477575

  11. Articular cartilage repair with autologous bone marrow mesenchymal cells.

    PubMed

    Matsumoto, Tomiya; Okabe, Takahiro; Ikawa, Tesshu; Iida, Takahiro; Yasuda, Hiroyuki; Nakamura, Hiroaki; Wakitani, Shigeyuki

    2010-11-01

    Articular cartilage defects that do not repair spontaneously induce osteoarthritic changes in joints over a long period of observation. In this study, we examined the usefulness of transplanting culture-expanded bone marrow mesenchymal cells into osteochondral defects of joints with cartilage defects. First, we performed experiments on rabbits and up on obtaining good results proceeded to perform the experiments on humans. Macroscopic and histological repair with this method was good, and good clinical results were obtained although there was no significant difference with the control group. Recent reports have indicated that this procedure is comparable to autologous chondrocyte implantation, and concluded that it was a good procedure because it required one step less than that required by surgery, reduced costs for patients, and minimized donor site morbidity. Although some reports have previously shown that progenitor cells formed a tumor when implanted into immune-deficient mice after long term in vitro culture, the safety of the cell transplantation was confirmed by our clinical experience. Thus, this procedure is useful, effective, and safe, but the repaired tissues were not always hyaline cartilage. To obtain better repair with this procedure, treatment approaches using some growth factors during in vitro culture or gene transfection are being explored.

  12. Evaluation of sodium alginate for bone marrow cell tissue engineering.

    PubMed

    Wang, L; Shelton, R M; Cooper, P R; Lawson, M; Triffitt, J T; Barralet, J E

    2003-09-01

    Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.

  13. Response and adaptation of bone cells to simulated microgravity

    NASA Astrophysics Data System (ADS)

    Hu, Lifang; Li, Runzhi; Su, Peihong; Arfat, Yasir; Zhang, Ge; Shang, Peng; Qian, Airong

    2014-11-01

    Bone loss induced by microgravity during space flight is one of the most deleterious factors on astronaut's health and is mainly attributed to an unbalance in the process of bone remodeling. Studies from the space microgravity have demonstrated that the disruption of bone remodeling is associated with the changes of four main functional bone cells, including osteoblast, osteoclast, osteocyte, and mesenchymal stem cells. For the limited availability, expensive costs and confined experiment conditions for conducting space microgravity studies, the mechanism of bone cells response and adaptation to microgravity is still unclear. Therefore, some ground-based simulated microgravity methods have been developed to investigate the bioeffects of microgravity and the mechanisms. Here, based on our studies and others, we review how bone cells (osteoblasts, osteoclasts, osteocytes and mesenchymal stem cells) respond and adapt to simulated microgravity.

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

    PubMed

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

    2015-01-01

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

  15. Unique cell culture systems for ground based research

    NASA Technical Reports Server (NTRS)

    Lewis, Marian L.

    1990-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2012-01-01

    Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 µA, were used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–materials interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 µA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 µA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell-materials interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. PMID:23144532

  18. THE EFFECT OF ANTISERUM, ALONE AND WITH HYDROCORTISONE, ON FOETAL MOUSE BONES IN CULTURE

    PubMed Central

    Fell, Honor B.; Weiss, L.

    1965-01-01

    1. The effects of normal rabbit serum and of rabbit antiserum to whole foetal mouse tissues, on the isolated limb bones of late foetal mice were studied in organ culture, and the influence of hydrocortisone on these effects was investigated. 2. Unheated normal serum caused slight loss of metachromatic material from the cartilage matrix, and some resorption of both cartilage and bone. 3. In unheated antiserum to foetal mouse tissues, the terminal cartilage was smaller and less metachromatic than in paired controls in normal serum, while osteoclasis was so intense that in many explants the bone had almost disappeared. The amount of necrosis varied with different batches of antiserum. 4. The changes produced by normal serum and antiserum could be largely prevented by heating the sera to 57°C for 45 minutes. 5. The effects could also be inhibited by the addition of hydrocortisone to the unheated sera; as little as 0.1 µg hydrocortisone per ml of medium had a well marked protective action. 6. It is suggested that (a) unheated antiserum causes a release of lysosomal enzymes with consequent breakdown of intercellular material, (b) this release is due to an indirect action on the lysosome via an increased permeability of the cell membrane, (c) hydrocortisone does not affect the antigen-antibody reaction, but inhibits the autolytic changes that normally follow this reaction, possibly by stabilising both the lysosomal and cell membranes. PMID:14276776

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

    PubMed Central

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

    2012-01-01

    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 κB 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. PMID:22265717

  20. Intramuscular injection of bone marrow mononuclear cells contributes to bone repair following midpalatal expansion in rats

    PubMed Central

    CHE, XIAOXIA; GUO, JIE; LI, XIANGDONG; WANG, LVE; WEI, SILONG

    2016-01-01

    Healing from injury requires the activation and proliferation of stem cells for tissue repair. Previous studies have demonstrated that bone marrow is a central pool of stem cells. The present study aimed to investigate the route undertaken by bone marrow mononuclear cells (BMMCs) following BMMC transplantation by masseter injection in a rat model of midpalatal expansion. The rats were divided into five groups according to the types of midpalatal expansion, incision and BMMC transplantation. Samples of midpalatal bone from the rats in each group were used for histological and immunohistochemical assessments to track and evaluate the differential potentials of the transplanted BMMCs in the masseter muscle and midpalatal bone. Bromodeoxyuridine was used as a BMMC tracing label, and M-cadherin was used to detect muscle satellite cells. The BMMCs injected into the masseter were observed, not only in the masseter, but also in the blood vessels and oral mucosa, and enveloped the midpalatal bone. A number of the BMMCs transformed into osteoblasts at the boundary of the neuromuscular bundle, and were embedded in the newly formed bone during midpalatal bone regeneration. The results of the present study suggested that BMMCs entered the circulation and migrated from muscle to the bone tissue, where they were involved in bone repair. Therefore, BMMCs may prove useful in the treatment of various types of cancer. PMID:26648442

  1. Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers.

    PubMed

    Chai, Yoke Chin; Roberts, Scott J; Desmet, Eline; Kerckhofs, Greet; van Gastel, Nick; Geris, Liesbet; Carmeliet, Geert; Schrooten, Jan; Luyten, Frank P

    2012-04-01

    Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (μCT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials. PMID:22269651

  2. Impaired Endothelial Progenitor Cell Mobilization and Dysfunctional Bone Marrow Stroma in Diabetes Mellitus

    PubMed Central

    Rafii, Shahin; Jaspers, Janneke E.; White, Ian A.; Hooper, Andrea T.; Doevendans, Pieter A.; Verhaar, Marianne C.

    2013-01-01

    Background Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired –at least partly– due to dysfunction of the bone marrow stromal compartment. Methods Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1+Flk-1+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34+ hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell–endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. Results In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. Conclusion EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients. PMID:23555959

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

    SciTech Connect

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

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first

  4. Placental-derived stem cells: Culture, differentiation and challenges

    PubMed Central

    Oliveira, Maira S; Barreto-Filho, João B

    2015-01-01

    Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

  5. 1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption.

    PubMed Central

    McSheehy, P M; Chambers, T J

    1987-01-01

    Although 1,25-dihydroxyvitamin D3 stimulates osteoclastic bone resorption in vivo and in organ culture, the mechanism by which it effects this stimulation is unknown. We have recently found that the agent does not stimulate resorption by osteoclasts mechanically disaggregated from bone and incubated on slices of cortical bone. This suggests that the osteoclasts were removed by disaggregation from the influence of some cell type, present in intact bone, that mediates hormone responsiveness. We therefore tested the ability of osteoblastic cells derived from neonatal rat calvariae and of cloned, hormone-responsive osteosarcoma cells (UMR106) to restore hormone responsiveness to unresponsive populations of osteoclasts. We found that osteoblastic cells from both sources induced a two- to fourfold stimulation of osteoclastic bone resorption in the presence of 1,25-dihydroxyvitamin D3. Stimulation was observed at concentrations of 10(-10) M and above. Actinomycin D and cycloheximide did not affect bone resorption by osteoclasts incubated alone, but abolished the capacity of osteoblastic cells to stimulate osteoclastic resorption in the presence of 1,25-dihydroxyvitamin D3. When calvarial cells or osteoblastlike UMR cells were incubated with the hormone, they produced a factor in cell-free supernatants that stimulated bone resorption by disaggregated osteoclasts. These experiments suggest that 1,25-dihydroxyvitamin D3 stimulates bone resorption through a primary action on osteoblastic cells, that are induced by the hormone to produce a factor that stimulates osteoclastic bone resorption. PMID:3611354

  6. Suppressor cells in transplantation tolerance. II. maturation of suppressor cells in the bone marrow chimera

    SciTech Connect

    Tutschka, P.J.; Ki, P.F.; Beschorner, W.E.; Hess, A.D.; Santos, G.W.

    1981-10-01

    Histoincompatible bone marrow allografts were established in lethally irradiated rats. At various times after transplantation, the spleen cells were harvested, subjected to mixed lymphocyte cultures, and assayed for suppressor cells in vitro and in vivo by adoptive transfer studies. Alloantigen-nonspecific suppressor cells appeared in the chimera at 40 days after grafting, coinciding with the resolution of graft-versus-host disease (GVHD). At 250 days the nonspecific suppressor cells were replaced by suppressor cells specifically suppressing donor-versus-host alloantigen responses. At 720 days suppressor cells could no longer be identified by in vitro methods but were identified by in vivo adoptive transfer of transplantation tolerance. After injection of host-type antigen into chimeras, the suppressor cells could be again demonstrated by in vitro methods.

  7. Suppressor cells in transplantation tolerance II. Maturation of suppressor cells in the bone marrow chimera

    SciTech Connect

    Tutschka, P.J.; Ki, P.F.; Beschorner, W.E.; Hess, A.D.; Santos, G.W.

    1981-10-01

    Histoincompatible bone marrow allografts were established in lethally irradiated rats. At various times after transplantation, the spleen cells were harvested, subjected to mixed lymphocyte cultures, and assayed for suppressor cells in vitro and in vivo by adoptive transfer studies. Alloantigen-nonspecific suppressor cells appeared in the chimera at 40 days after grafting, coinciding with the resolution of graft-versus-host disease (GVHD). At 250 days the nonspecific suppressor cells were replaced by suppressor cells specifically suppressing donor-versus-host alloantigen responses. At 720 days suppressor cells could no longer be identified by in vitro methods but were identified by in vivo adoptive transfer of transplantation tolerance. After injection of host-type antigen into chimeras, the suppressor cells could be again demonstrated by in vitro methods.

  8. Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow.

    PubMed

    Montali, Marina; Barachini, Serena; Pacini, Simone; Panvini, Francesca M; Petrini, Mario

    2016-01-01

    In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells". PMID:27500428

  9. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro.

    PubMed

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N; Neutzsky-Wulff, Anita V; Geisler, Carsten; Karlsson, Lars; Söderström, Kalle

    2015-07-01

    Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions.

  10. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro

    PubMed Central

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N; Neutzsky-Wulff, Anita V; Geisler, Carsten; Karlsson, Lars; Söderström, Kalle

    2015-01-01

    Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions. PMID:25684021

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

    PubMed

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

    2015-01-01

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

  12. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

    SciTech Connect

    Tu Qisheng; Valverde, Paloma . E-mail: paloma.valverde@tufts.edu; Chen, Jake

    2006-03-24

    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.

  13. Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects

    PubMed Central

    Ode, G.; Hoelscher, G.; Ingram, J.; Bethea, S.; Bosse, M. J.

    2016-01-01

    Objectives The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics. Methods Following Institutional Review Board approval, biomembrane specimens were obtained from 12 patient surgeries for management of segmental bony defects (mean patient age 40.7 years, standard deviation 14.4). Biomembranes from nine tibias and three femurs were processed for morphologic, molecular or stem cell analyses. Gene expression was determined using the Affymetrix GeneChip Operating Software (GCOS). Molecular analyses compared biomembrane gene expression patterns with a mineralising osteoblast culture, and gene expression in specimens with longer spacer duration (> 12 weeks) with specimens with shorter durations. Statistical analyses used the unpaired student t-test (two tailed; p < 0.05 was considered significant). Results Average PMMA spacer in vivo time was 11.9 weeks (six to 18). Trabecular bone was present in 33.3% of the biomembrane specimens; bone presence did not correlate with spacer duration. Biomembrane morphology showed high vascularity and collagen content and positive staining for the key bone forming regulators, bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). Positive differentiation of cultured biomembrane cells for osteogenesis was found in cells from patients with PMMA present for six to 17 weeks. Stem cell differentiation showed greater variability in pluripotency for osteogenic potential (70.0%) compared with chondrogenic or adipogenic potentials (100% and 90.0%, respectively). Significant upregulation of BMP2 and 6, numerous collagens, and bone gla protein was present in biomembrane compared with the cultured cell line. Biomembranes with

  14. Human Bone Marrow Stromal Cells: A Reliable, Challenging Tool for In Vitro Osteogenesis and Bone Tissue Engineering Approaches.

    PubMed

    Hempel, Ute; Müller, Katrin; Preissler, Carolin; Noack, Carolin; Boxberger, Sabine; Dieter, Peter; Bornhäuser, Martin; Wobus, Manja

    2016-01-01

    Adult human bone marrow stromal cells (hBMSC) are important for many scientific purposes because of their multipotency, availability, and relatively easy handling. They are frequently used to study osteogenesis in vitro. Most commonly, hBMSC are isolated from bone marrow aspirates collected in clinical routine and cultured under the "aspect plastic adherence" without any further selection. Owing to the random donor population, they show a broad heterogeneity. Here, the osteogenic differentiation potential of 531 hBMSC was analyzed. The data were supplied to correlation analysis involving donor age, gender, and body mass index. hBMSC preparations were characterized as follows: (a) how many passages the osteogenic characteristics are stable in and (b) the influence of supplements and culture duration on osteogenic parameters (tissue nonspecific alkaline phosphatase (TNAP), octamer binding transcription factor 4, core-binding factor alpha-1, parathyroid hormone receptor, bone gla protein, and peroxisome proliferator-activated protein γ). The results show that no strong prediction could be made from donor data to the osteogenic differentiation potential; only the ratio of induced TNAP to endogenous TNAP could be a reliable criterion. The results give evidence that hBMSC cultures are stable until passage 7 without substantial loss of differentiation potential and that established differentiation protocols lead to osteoblast-like cells but not to fully authentic osteoblasts. PMID:27293446

  15. Human Bone Marrow Stromal Cells: A Reliable, Challenging Tool for In Vitro Osteogenesis and Bone Tissue Engineering Approaches

    PubMed Central

    Hempel, Ute; Müller, Katrin; Preissler, Carolin; Noack, Carolin; Boxberger, Sabine; Dieter, Peter; Bornhäuser, Martin; Wobus, Manja

    2016-01-01

    Adult human bone marrow stromal cells (hBMSC) are important for many scientific purposes because of their multipotency, availability, and relatively easy handling. They are frequently used to study osteogenesis in vitro. Most commonly, hBMSC are isolated from bone marrow aspirates collected in clinical routine and cultured under the “aspect plastic adherence” without any further selection. Owing to the random donor population, they show a broad heterogeneity. Here, the osteogenic differentiation potential of 531 hBMSC was analyzed. The data were supplied to correlation analysis involving donor age, gender, and body mass index. hBMSC preparations were characterized as follows: (a) how many passages the osteogenic characteristics are stable in and (b) the influence of supplements and culture duration on osteogenic parameters (tissue nonspecific alkaline phosphatase (TNAP), octamer binding transcription factor 4, core-binding factor alpha-1, parathyroid hormone receptor, bone gla protein, and peroxisome proliferator-activated protein γ). The results show that no strong prediction could be made from donor data to the osteogenic differentiation potential; only the ratio of induced TNAP to endogenous TNAP could be a reliable criterion. The results give evidence that hBMSC cultures are stable until passage 7 without substantial loss of differentiation potential and that established differentiation protocols lead to osteoblast-like cells but not to fully authentic osteoblasts. PMID:27293446

  16. Temporal bone squamous cell carcinoma - Penang experience.

    PubMed

    Ng, S Y; Pua, K C; Zahirrudin, Z

    2015-12-01

    Temporal bone squamous cell carcinoma (TBSCC) is rare and poses difficulties in diagnosing, staging and management. We describe a case series with six patients who were diagnosed TBSCC, from January 2009 to June 2014, with median age of 62 years old. All patients presented with blood-stain discharge and external auditory canal mass, showing that these findings should highly alert the diagnosis of TBSCC. Three patients staged T3 and another three with T4 disease. High-resolution CT (HRCT) temporal findings were noted to be different from intraoperative findings and therefore we conclude that MRI should be done to look for middle ear involvement or other soft tissue invasion for more accurate staging. Lateral temporal bone resection (LTBR) and parotidectomy was done for four patients with or without neck dissection. Patients with positive margin, perineural invasion or parotid and glenoid involvement carry poorer prognosis and postoperative radiotherapy may improve the survival rate. One patient had successful tumor resection via piecemeal removal approach in contrast with the recommended en bloc resection shows that with negative margin achieved, piecemeal removal approach can be a good option for patients with T2-3 disease. In general, T4 tumor has dismal outcome regardless of surgery or radiotherapy given.

  17. Ultrastructural and mineral phase characterization of the bone-like matrix assembled in F-OST osteoblast cultures.

    PubMed

    Querido, W; Abraçado, L G; Rossi, A L; Campos, A P C; Rossi, A M; San Gil, R A S; Borojevic, R; Balduino, A; Farina, M

    2011-11-01

    Cell cultures are often used to study bone mineralization; however, not all systems achieve a bone-like matrix formation. In this study, the mineralized matrix assembled in F-OST osteoblast cultures was analyzed, with the aim of establishing a novel model for bone mineralization. The ultrastructure of the cultures was investigated using scanning electron microscopy, atomic force microscopy, and transmission electron microscopy (TEM). The mineral phase was characterized using conventional and high-resolution TEM, energy-dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and solid-state (31)P and (1)H nuclear magnetic resonance. F-OST osteoblast cultures presented a clear nodular mineralization pattern. The chief features of the mineralizing nodules were globular accretions ranging from about 100 nm to 1.5 μm in diameter, loaded with needle-shaped crystallites. Accretions seemed to bud from the cell membrane, increase in size, and coalesce into larger ones. Arrays of loosely packed, randomly oriented collagen fibrils were seen along with the accretions. Mineralized fibrils were often observed, sometimes in close association with accretions. The mineral phase was characterized as a poorly crystalline hydroxyapatite. The Ca/P atomic ratio was 1.49 ± 0.06. The presence of OH was evident. The lattice parameters were a = 9.435 Å and c = 6.860 Å. The average crystallite size was 20 nm long and 10 nm wide. Carbonate substitutions were seen in phosphate and OH sites. Water was also found within the apatitic core. In conclusion, F-OST osteoblast cultures produce a bone-like matrix and may provide a good model for bone mineralization studies.

  18. Mesenchymal progenitor cells in red and yellow bone marrow.

    PubMed

    Gurevitch, O; Slavin, S; Resnick, I; Khitrin, S; Feldman, A

    2009-01-01

    Marrow cavities in all bones of newborn mammals contain haematopoietic tissue and stromal microenvironment that support haematopoiesis (haematopoietic microenvironment), known as red bone marrow (BM). From the early postnatal period onwards, the haematopoietic microenvironment, mainly in tubular bones of the extremities, is replaced by mesenchymal cells that accumulate lipid drops, known as yellow BM, whereas haematopoietic tissue gradually disappears. We analysed the ability of mesenchymal cell progenitors in red and yellow BM to produce bone and haematopoietic microenvironment in vivo after transplantation into normal or haematopoietically deficient (irradiated and old) recipients. We found that (1) normal substitution of red with yellow BM results from a gradual loss of mesenchymal stem cells (MSCs) capable of developing bone and haematopoietic microenvironment; (2) the mesenchymal cell population in tubular bones still containing active haematopoietic tissue gradually becomes depleted of MSCs, starting from a young age; (3) haematopoietic microenvironment is incapable of self-maintenance and its renewal depends on the presence of precursor cells; (4) the mesenchymal cell population remaining in areas with yellow BM contains cells able to develop functionally active haematopoietic microenvironment in conditions of haematopoietic insufficiency. Our data also indicate the possible existence of bi-potential stromal precursor cells producing either bone in normal, or bone together with active haematopoietic microenvironment in irradiated or old recipients. This study opens a spectrum of opportunities for the extension of haematopoietic territories by substituting the fat contents of BM cavities with haematopoietic tissue, thereby improving haematopoiesis compromised by cytotoxic treatments, irradiation, ageing, etc.

  19. Bone reconstruction of large defects using bone marrow derived autologous stem cells.

    PubMed

    Lucarelli, Enrico; Donati, Davide; Cenacchi, Annarita; Fornasari, Pier Maria

    2004-04-01

    Bone is a tissue that has the ability to heal itself when fractured. Occasionally, a critical defect can be formed when part of the bone is lost or excised, in this case the bone fails to heal and requires bone reconstruction to prevent a non-union defect. Autogenous cancellous bone is the current gold standard treatment in bone loss. Because the amount of autogenous cancellous bone that can be harvested is limited, the expanding need for bone reconstruction is paired by the growth of interest in the discipline of tissue engineering. Labs worldwide are working to provide the right carrier and the right set of cells that, once retransplanted, will ensure bone repair. Several investigators have focused their attention on a subset of autologous non-hematopoietic stem/progenitor cells contained in the adult bone marrow stroma, referred to as stromal stem cells (SSC), as the appropriate cells to be transplanted. The use of autologous cells is facilitated by less stringent ethical and regulatory issues and does not require the patient to be immunologically suppressed. In pre-clinical and clinical protocols of critical defects in which SSC are employed, two approaches are mainly used: in the first, SSC are derived from bone marrow and directly introduced at the lesion site, in the second, SSC are derived from several sites and are expanded ex vivo before being implanted. Both approaches, equally correct in principle, will have to demonstrate, with definitive evidence of their efficacy, their capability of solving a critical clinical problem such as non-union. In this report we outline the difficulties of working with SSC.

  20. The Challenge and the Promise of Bone Marrow Cells for Human Cartilage Repair

    PubMed Central

    2015-01-01

    The cartilage repair potential of bone marrow–derived stem cells has been well described. Harnessing this potential for human articular cartilage repair remains challenging. Accessing bone marrow repair cells through marrow stimulation techniques such as microfracture is readily achieved with generally good but inconsistent results. Animal and human studies show feasibility for ex vivo processing of bone marrow to isolate, concentrate, and culture mesenchymal stem cells. Nevertheless, it has been difficult to show consistent and clinically meaningful improvement using bone marrow cell preparations above what has been achieved with microfracture. Consequently, microfracture continues to be the simplest and most commonly used method to enhance repair of focal articular cartilage defects. Emerging preclinical work in the equine model suggests a role for enhancing marrow-stimulation techniques through the use of natural scaffolds such as autologous platelet enriched fibrin as well as optimization of joint biology through localized gene therapy to support cartilage repair. In contrast to joint replacement where inert materials of known mechanical properties are used, host biology determines the relative success, failure, and durability of cartilage repair. As such, development of personalized strategies to improve the quality and durability of bone marrow cell–based articular cartilage repair represent exciting new areas of inquiry. Continued advances in stem cell biology, scaffold technologies, and methods to delineate and enhance host biology, both systemically and within the joint, hold promise for harnessing the full power of bone marrow cells to facilitate cartilage repair and regeneration. PMID:27340515

  1. Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages

    PubMed Central

    2014-01-01

    Background The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Results Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Conclusion Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent. PMID:25281406

  2. Integrins and bone metastasis: integrating tumor cell and stromal cell interactions.

    PubMed

    Schneider, Jochen G; Amend, Sarah R; Weilbaecher, Katherine N

    2011-01-01

    Integrins on both tumor cells and the supporting host stromal cells in bone (osteoclasts, new blood vessels, inflammatory cells, platelets and bone marrow stromal cells) play key roles in enhancing bone metastasis. Tumor cells localize to specific tissues through integrin-mediated contacts with extracellular matrix and stromal cells. Integrin expression and signaling are perturbed in cancer cells, allowing them to "escape" from cell-cell and cell-matrix tethers, invade, migrate and colonize within new tissues and matrices. Integrin signaling through αvβ3 and VLA-4 on tumor cells can promote tumor metastasis to and proliferation in the bone microenvironment. Osteoclast (OC) mediated bone resorption is a critical component of bone metastasis and can promote tumor growth in bone and αvβ3 integrins are critical to OC function and development. Tumors in the bone microenvironment can recruit new blood vessel formation, platelets, pro-tumor immune cells and bone marrow stromal cells that promote tumor growth and invasion in bone. Integrins and their ligands play critical roles in platelet aggregation (αvβ3 and αIIbβ3), hematopoietic cell mobilization (VLA-4 and osteopontin), neoangiogenesis (αvβ3, αvβ5, α6β4, and β1 integrin) and stromal function (osteopontin and VLA-4). Integrins are involved in the pathogenesis of bone metastasis at many levels and further study to define integrin dysregulation by cancer will yield new therapeutic targets for the prevention and treatment of bone metastasis.

  3. Effects of alendronate and pamidronate on cultured rat metatarsal bones: failure to prevent dexamethasone-induced growth retardation.

    PubMed

    Heino, Terhi J; Chagin, Andrei S; Takigawa, Masaharu; Sävendahl, Lars

    2008-04-01

    Bisphosphonates are widely used anti-resorptive drugs in the adult population. In children, their use has mainly been limited to patients with osteogenesis imperfecta. However, the powerful effects of bisphosphonates on bone turnover have raised concern about their long-term effects on the growing skeleton. We aimed to study the effects of two commonly used bisphosphonates, alendronate (Aln) and pamidronate (Pam) on normal bone growth as well as their potential to prevent glucocorticoid-induced growth retardation. Effects on bone growth were studied in fetal rat metatarsal bones (day E20) that were cultured for 5-47 days and measured every 2-7 days. Cellular mechanisms were investigated in metatarsal bones and also in the human chondrocytic cell line HCS-2/8. Chondrocyte viability (WST-1), proliferation (BrdU incorporation), differentiation (collagen type X immunohistochemistry) and apoptosis (TUNEL and Cell Death ELISA) were determined. At a clinically relevant concentration of bisphosphonates (1 microM), metatarsal bone growth was stimulated by both Aln (p<0.001 for length and p<0.05 for width) and Pam (p<0.05 for both length and width) from day 19 of culture. The growth-stimulatory effect was associated with increased chondrocyte proliferation (+21% with Aln and +24% with Pam), while cell differentiation and apoptosis were not affected. Despite the finding that both Aln and Pam (1 muM) rescued HCS-2/8 cells from undergoing dexamethasone-induced apoptosis, neither of them was able to prevent dexamethasone-induced growth retardation of fetal rat metatarsal bones. Aln and Pam have the capacity to stimulate the growth of cultured fetal rat metatarsal bones; an effect associated with increased proliferation of growth plate chondrocytes. Our experimental data suggest that bisphosphonates are ineffective in preventing glucocorticoid-induced growth retardation. Nevertheless, based on our in vitro data, both Aln and Pam appear safe to use in growing children, at least

  4. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.

    PubMed

    Hudu, Shuaibu Abdullahi; Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-03-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent. PMID:27134874

  5. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases

    PubMed Central

    Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-01-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the “gold standard” for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent. PMID:27134874

  6. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.

    PubMed

    Hudu, Shuaibu Abdullahi; Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-03-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent.

  7. Mesenchymal stem cells: mechanisms and role in bone regeneration

    PubMed Central

    Qin, Yunhao; Guan, Junjie; Zhang, Changqing

    2014-01-01

    Stimulating bone growth and regeneration, especially in patients with delayed union or non-union of bone, is a challenge for orthopaedic surgeons. Treatments employed for bone regeneration are based on the use of cells, biomaterials and factors. Among these therapies, cell treatment with mesenchymal stem cells (MSCs) has a number of advantages as MSCs: (1) are multipotent cells that can migrate to sites of injury; (2) are capable of suppressing the local immune response; and (3) are available in large quantities from the patients themselves. MSC therapies have been used for stimulating bone regeneration in animal models and in patients. Methods of application range from direct MSC injection, seeding MSCs on synthetic scaffolds, the use of gene-modified MSCs, and hetero-MSCs application. However, only a small number of these cell-based strategies are in clinical use, and none of these treatments has become the gold standard treatment for delayed or non-union of bone. PMID:25335795

  8. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  9. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  10. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  11. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  12. 9 CFR 101.6 - Cell cultures.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  13. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-11-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18428384

  14. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18265370

  15. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18770828

  16. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-12-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18429293

  17. Therapeutic effect of bone marrow mesenchymal stem cells on cold stress induced changes in the hippocampus of rats.

    PubMed

    Kumar, Saravana Kumar Sampath; Perumal, Saraswathi; Rajagopalan, Vijayaraghavan

    2014-10-01

    The present study aims to evaluate the effect of bone marrow mesenchymal stem cells on cold stress induced neuronal changes in hippocampal CA1 region of Wistar rats. Bone marrow mesenchymal stem cells were isolated from a 6-week-old Wistar rat. Bone marrow from adult femora and tibia was collected and mesenchymal stem cells were cultured in minimal essential medium containing 10% heat-inactivated fetal bovine serum and were sub-cultured. Passage 3 cells were analyzed by flow cytometry for positive expression of CD44 and CD90 and negative expression of CD45. Once CD44 and CD90 positive expression was achieved, the cells were cultured again to 90% confluence for later experiments. Twenty-four rats aged 8 weeks old were randomly and evenly divided into normal control, cold water swim stress (cold stress), cold stress + PBS (intravenous infusion), and cold stress + bone marrow mesenchymal stem cells (1 × 10(6); intravenous infusion) groups. The total period of study was 60 days which included 1 month stress period followed by 1 month treatment. Behavioral functional test was performed during the entire study period. After treatment, rats were sacrificed for histological studies. Treatment with bone marrow mesenchymal stem cells significantly increased the number of neuronal cells in hippocampal CA1 region. Adult bone marrow mesenchymal stem cells injected by intravenous administration show potential therapeutic effects in cognitive decline associated with stress-related lesions.

  18. Osteoclast-independent bone resorption by fibroblast-like cells

    PubMed Central

    Pap, Thomas; Claus, Anja; Ohtsu, Susumu; Hummel, Klaus M; Schwartz, Peter; Drynda, Susanne; Pap, Géza; Machner, Andreas; Stein, Bernhard; George, Michael; Gay, Renate E; Neumann, Wolfram; Gay, Steffen; Aicher, Wilhelm K

    2003-01-01

    To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders. PMID:12723988

  19. Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate.

    PubMed

    Corradetti, Bruna; Taraballi, Francesca; Powell, Sebastian; Sung, David; Minardi, Silvia; Ferrari, Mauro; Weiner, Bradley K; Tasciotti, Ennio

    2015-05-01

    Bone is a dynamic organ where skeletal progenitors and hematopoietic cells share and compete for space. Presumptive mesenchymal stem cells (MSC) have been identified and harvested from the bone marrow (BM-MSC) and cortical bone fragments (CBF-MSC). In this study, we demonstrate that despite the cells sharing a common ancestor, the differences in the structural properties of the resident tissues affect cell behavior and prime them to react differently to stimuli. Similarly to the bone marrow, the cortical portion of the bone contains a unique subset of cells that stains positively for the common MSC-associated markers. These cells display different multipotent differentiation capability, clonogenic expansion, and immunosuppressive potential. In particular, when compared with BM-MSC, CBF-MSC are bigger in size, show a lower proliferation rate at early passages, have a greater commitment toward the osteogenic lineage, constitutively produce nitric oxide as a mediator for bone remodeling, and more readily respond to proinflammatory cytokines. Our data suggest that the effect of the tissue's microenvironment makes the CBF-MSC a superior candidate in the development of new strategies for bone repair.

  20. [The application progress of human urine derived stem cells in bone tissue engineering].

    PubMed

    Gao, Peng; Jiang, Dapeng; Li, Zhaozhu

    2016-04-01

    The research of bone tissue engineering bases on three basic directions of seed cells, scaffold materials and growth information. Stem cells have been widely studied as seed cells. Human urine-derived stem cell (hUSC) is extracted from urine and described to be adhesion growth, cloning, expression of the majority of mesenchymal stem cell markers and peripheral cell markers, multi-potential and no tumor but stable karyotype with passaging many times. Some researches proposed that hUSC might be a new source of seed cells in tissue engineering because of their invasive and convenient obtention, stable culture and multiple differentiation potential. PMID:27029208

  1. Interleukin-1 and tumor necrosis factor-alpha induce collagenolysis and bone resorption by regulation of matrix metalloproteinase-2 in mouse calvarial bone cells.

    PubMed

    Kang, Bong-Seok; Park, Young-Guk; Cho, Jin-Young; Kim, June-Ki; Lee, Tae-Kyun; Kim, Dong-Wook; Gu, Yeun-Hwa; Suzuki, Ikukatsu; Chang, Young-Chae; Kim, Cheorl-Ho

    2003-08-01

    Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) greatly induces osteoclast formation and stimulates bone resorption of mouse calvaria in culture. We examined the effects of the two cytokines on the collagenolysis and bone resorption by induction of matrix metalloproteinases (MMPs). The cells were analyzed using zymographic analysis. It was shown that the mouse calvarial osteoblasts constitutively synthesize progelatinase-A (MMP-2). Interleukin-1beta markedly enhanced the messenger RNAs (mRNAs) expression of MMP-2 (gelatinase A), but slightly MMP-9 (gelatinase B), which associated with increases in bone matrix degradation. Both pro- and active-forms of MMP-2 were detected in the conditioned medium collected from calvarial cultures, and IL-1beta markedly stimulated both pro- and active-forms of the MMP-2. The expression of MMP-2 mRNAs could be detected, and they were markedly enhanced by IL-1beta on days 1 and 2. These results demonstrate that the potency of induction of MMP-2 by IL-1beta and TNF-alpha is closely linked to the respective bone-resorbing activity, suggesting that MMP-2-dependent degradation of bone matrix plays a key role in bone resorption induced by these cytokines. On the other hand, when the mouse osteoblasts were stimulated with parathyroid hormone, 1,25(OH)2D3, mononuclear cell conditioned medium (MCM) and IL-1 as bone resorption agents, collagenolysis was increased by producing the active gelatinase. Interleukin-1 in stimulating bone resorption was examined using fetal mouse long bone organ culture. Interleukin-1 stimulated bone resorption and produced marked resorption when present simultaneously. Furthermore, treatment of indomethacin and dexamethasone clearly abolished the responses of IL-1alpha and IL-1beta.

  2. Cultivation of Human Bone-Like Tissue from Pluripotent Stem Cell-Derived Osteogenic Progenitors in Perfusion Bioreactors

    PubMed Central

    de Peppo, Giuseppe Maria; Vunjak-Novakovic, Gordana; Marolt, Darja

    2014-01-01

    Human pluripotent stem cells represent an unlimited source of skeletal tissue progenitors for studies of bone biology, pathogenesis, and the development of new approaches for bone reconstruction and therapies. In order to construct in vitro models of bone tissue development and to grow functional, clinical-size bone substitutes for transplantation, cell cultivation in three-dimensional environments composed of porous osteoconductive scaffolds and dynamic culture systems—bioreactors—has been studied. Here, we describe a stepwise procedure for the induction of human embryonic and induced pluripotent stem cells (collectively termed PSCs) into mesenchymal-like progenitors, and their subsequent cultivation on decellularized bovine bone scaffolds in perfusion bioreactors, to support the development of viable, stable bone-like tissue in defined geometries. PMID:24281874

  3. Multistage carcinogenesis in cell culture.

    PubMed

    Rubin, H

    2001-01-01

    Rodent fibroblasts explanted from embryos to culture undergo a period of declining growth rate in serial passages leading to crisis, followed by the appearance of variants which can multiply indefinitely. If the "immortal" cell line was established by low density passage, i.e., 3T3 cells, it has a low saturation density and is non-tumorigenic. If it was established by high density passage, it has a high saturation density and is tumorigenic. The establishment of cells goes through successive stages, including increased capacity to multiply in low serum concentration, growth to high saturation density, growth in suspension, assisted tumour formation in susceptible hosts and unassisted tumour formation. Chromosome aberrations and aneuploidy occur long before the capacity to produce tumours appears. Contrary to conventional belief, human fibroblast populations also undergo a continuous loss of capacity to multiply from the time of explantation, with only the longest surviving clone reaching the Hayflick limit. Neoplastic transformation of rodent cells is strongly favoured by maintaining them in a quiescent state at confluence for prolonged periods, which results in genetic damage to the cells. It also produces a large variety of chromosomal aberrations in human cells and extends their replicative lifespan. Individual clones are more susceptible to spontaneous transformation than their heterogeneous parental cultures. The implications of these results for tumour development in vivo are that oncogenic genetic changes may be common under stressful conditions which restrict replication, and that such changes are maximized when a rogue clone reaches a critical size that reduces stabilizing interactions with neighbouring clones. An alternative explanation, described in the Addendum, which we retrospectively favor is that the easily transformed clones are a minority in the uncloned parental population. The reason they transform before the parental population is that when

  4. Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics.

    PubMed

    N'diaye, Marie; Warnecke, Andreas; Flytzani, Sevasti; Abdelmagid, Nada; Ruhrmann, Sabrina; Olsson, Tomas; Jagodic, Maja; Harris, Robert A; Guerreiro-Cacais, Andre Ortlieb

    2016-03-01

    Dendritic cells are professional APCs that play a central role in the initiation of immune responses. The limited ex vivo availability of dendritic cells inspires the widespread use of bone marrow-derived dendritic cells as an alternative in research. However, the functional characteristics of bone marrow-derived dendritic cells are incompletely understood. Therefore, we compared functional and phenotypic characteristics of rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3 ligand bone marrow-derived dendritic cells. A comparison of surface markers revealed that FLT3 ligand-bone marrow-derived dendritic cells expressed signal regulatory protein α, CD103, and CD4 and baseline levels of MHC class II, CD40, and CD86, which were highly up-regulated upon stimulation. Conversely, GM-CSF/IL-4-bone marrow-derived dendritic cells constitutively expressed signal regulatory protein α, CD11c, and CD11b but only mildly up-regulated MHC class II, CD40, or CD86 following stimulation. Expression of dendritic cell-associated core transcripts was restricted to FLT3 ligand-bone marrow-derived dendritic cells . GM-CSF/IL-4-bone marrow-derived dendritic cells were superior at phagocytosis but were outperformed by FLT3 ligand-bone marrow-derived dendritic cells at antigen presentation and T cell stimulation in vitro. Stimulated GM-CSF/IL-4-bone marrow-derived dendritic cells secreted more TNF, CCL5, CCL20, and NO, whereas FLT3 ligand-bone marrow-derived dendritic cells secreted more IL-6 and IL-12. Finally, whereas GM-CSF/IL-4-bone marrow-derived dendritic cell culture supernatants added to resting T cell cultures promoted forkhead box p3(+) regulatory T cell populations, FLT3 ligand-bone marrow-derived dendritic cell culture supernatants drove Th17 differentiation. We conclude that rat GM-CSF/IL-4-bone marrow-derived dendritic cells and FLT3 ligand-bone marrow-derived dendritic cells are functionally distinct. Our data support the current rationale that FLT3

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

    PubMed Central

    Hombauer, H; Minguell, J J

    2000-01-01

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

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

    PubMed Central

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

    2014-01-01

    Objective: Evaluate the behavior and function of human umbilical vein endothelial cells (HUVECs) on decellularized extracellular matrix (ECM) deposited by bone marrow mesenchymal stem cells (BMSCs). Methods: Prepared through chemical approach, decellularized ECM was characterized by use of immunofluorescence staining. The morphology, attachment, proliferation and migration of HUVECs cultured on six-well tissue culture plastic (TCP) and decellularized ECM were investigated. Results: Decellularized ECM was successfully prepared without three-dimensional architecture disruption. This biological scaffold is similar to nature vascular ECM, preserved various matrix proteins such as type I collagen, type III collagen and fibronection. HUVECs on decellularized ECM showed well attachment and regular arrangement. Decellularized ECM could also significantly enhance the migration and proliferation potential of HUVECs in contrast to TCP. Conclusion: Deposited by BMSCs, ECM can affect the behavior of endothelial cell and could be used as a promising material in tissue engineering. PMID:25663998

  7. The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts

    PubMed Central

    ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I.; Davison, Noel L.; de Vries, Teun J.; Everts, Vincent

    2015-01-01

    Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones—cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme

  8. Osteoblastic Wnts differentially regulate bone remodeling and the maintenance of bone marrow mesenchymal stem cells.

    PubMed

    Wan, Yong; Lu, Cheng; Cao, Jingjing; Zhou, Rujiang; Yao, Yiyun; Yu, Jian; Zhang, Lingling; Zhao, Haixia; Li, Hanjun; Zhao, Jianzhi; Zhu, Xuming; He, Lin; Liu, Yongzhong; Yao, Zhengju; Yang, Xiao; Guo, Xizhi

    2013-07-01

    Wnt signaling has important roles in embryonic bone development and postnatal bone remodeling, but inconsistent impact on bone property is observed in different genetic alterations of Lrp5 and β-catenin. More importantly, it is still controversial whether Lrp5 regulate bone formation locally or globally through gut-derived serotonin. Here we explored the function of Wnt proteins in osteoblastic niche through inactivation of the Wntless (Wls) gene, which abrogates the secretion of Wnts. The depletion of Wls in osteoblast progenitor cells resulted in severe osteopenia with more profound defects in osteoblastogenesis, osteoclastogenesis and maintenance of bone marrow mesenchymal stem cells (BMSCs) compared to that observed in Lrp5 and β-catenin mutants. These findings support the point of view that Wnt/Lrp5 signaling locally regulates bone mass accrual through multiple effects of osteoblastic Wnts on osteoblastic bone formation and osteoclastic bone resorption. Moreover, osteoblastic Wnts confer a niche role for maintenance of BMSCs, providing novel cues for the definition of BMSCs niche in bone marrow.

  9. Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids.

    PubMed

    Suenaga, Hideyuki; Furukawa, Katsuko S; Suzuki, Yukako; Takato, Tsuyoshi; Ushida, Takashi

    2015-11-01

    Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, β-tricalcium phosphate (β-TCP) granules, or β-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60% those of native bones, respectively). All tests showed poor bone regeneration in the β-TCP and β-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

  10. Basic techniques in mammalian cell tissue culture.

    PubMed

    Phelan, Katy; May, Kristin M

    2015-03-02

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.

  11. Bone marrow processing for transplantation using Cobe Spectra cell separator.

    PubMed

    Veljković, Dobrila; Nonković, Olivera Šerbić; Radonjić, Zorica; Kuzmanović, Miloš; Zečević, Zeljko

    2013-06-01

    Concentration of bone marrow aspirates is an important prerequisite prior to infusion of ABO incompatible allogeneic marrow and prior to cryopreservation and storage of autologous marrow. In this paper we present our experience in processing 15 harvested bone marrow for ABO incompatible allogeneic and autologous bone marrow (BM) transplantation using Cobe Spectra® cell separator. BM processing resulted in the median recovery of 91.5% CD34+ cells, erythrocyte depletion of 91% and volume reduction of 81%. BM processing using cell separator is safe and effective technique providing high rate of erythrocyte depletion and volume reduction, and acceptable recovery of the CD34+ cells.

  12. Resveratrol Increases the Bone Marrow Hematopoietic Stem and Progenitor Cell Capacity

    PubMed Central

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

    2014-01-01

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

  13. Bone grafts engineered from human adipose-derived stem cells in dynamic 3D-environments.

    PubMed

    Declercq, Heidi A; De Caluwé, Tamara; Krysko, Olga; Bachert, Claus; Cornelissen, Maria J

    2013-01-01

    Modular tissue engineering (TE) is a promising alternative to overcome the limits in traditional TE. In the present study, adipose tissue derived stem cells (ADSC)-laden microcarriers are used as building blocks (microtissues) that self-assemble into macrotissues in a bottom-up approach. These bone grafts were compared with a classical top-down approach (scaffolds). This concept was compared with bone marrow derived stem cells (BMSC) as cell source. Cells were immunophenotypically analyzed, followed by 2D/3D osteogenic differentiation in static/dynamic conditions. The bone graft quality was evaluated by (immuno)histochemistry and gene expression. After 6 weeks of dynamic culturing, scaffolds were highly colonized although not in the center and the osteogenic gene expression was higher in contrast to static cultures. A cell-to-microcarrier ratio of 5 × 10(6) cells-0.09 g microcarriers leaded to aggregate formation resulting in microtissues with subsequent macrotissue formation. ADSC/BMSC on scaffolds showed a downregulation of Runx2 and collagen I, demonstrating the end-stage, in contrary to microcarriers, where an upregulation of Runx2, collagen I together with BSP and osteocalcin was observed. This paper showed that high quality bone grafts (2 cm³) can be engineered in a bottom-up approach with cell-laden microcarriers.

  14. Dynamized preparations in cell culture.

    PubMed

    Sunila, Ellanzhiyil Surendran; Kuttan, Ramadasan; Preethi, Korengath Chandran; Kuttan, Girija

    2009-06-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  15. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow

    SciTech Connect

    Itoh, K.; Tezuka, H.; Sakoda, H.; Konno, M.; Nagata, K.; Uchiyama, T.; Uchino, H.; Mori, K.J.

    1989-02-01

    Stromal cell lines, designated MS-1, -2, -3, -4, -5, -6, and -7 were established by irradiating the adherent cells in long-term bone marrow cultures with 900-rad x-rays. Two of the cell lines, MS-1 and MS-5, have the capacity to support the growth of hemopoietic stem cells (spleen colony-forming cells and granulocyte-macrophage colony-forming cells) for greater than 2 months in vitro. These two cell lines were alkaline phosphatase-, peroxidase-, and factor VIII-negative and positive for periodic acid-Schiff and nonspecific esterase. Extracellular matrix proteins such as fibronectin, laminin, and collagen type I were produced by these two cell lines. Neither MS-1 cell- nor MS-5 cell-conditioned medium supported the growth of hemopoietic stem cells, and hemopoietic stem cells were found preferentially to be under and on MS-1 and MS-5 layers rather than in suspension. Close contact with the MS-1 cell layer or the MS-5 cell layer appears to be essential in maintaining hemopoiesis in vitro. Conditioned media from MS-1 cells and MS-5 cells stimulated granulocyte colony formation from murine bone marrow cells in semisolid culture.

  16. Stem and progenitor cells: advancing bone tissue engineering.

    PubMed

    Tevlin, R; Walmsley, G G; Marecic, O; Hu, Michael S; Wan, D C; Longaker, M T

    2016-04-01

    Unlike many other postnatal tissues, bone can regenerate and repair itself; nevertheless, this capacity can be overcome. Traditionally, surgical reconstructive strategies have implemented autologous, allogeneic, and prosthetic materials. Autologous bone--the best option--is limited in supply and also mandates an additional surgical procedure. In regenerative tissue engineering, there are myriad issues to consider in the creation of a functional, implantable replacement tissue. Importantly, there must exist an easily accessible, abundant cell source with the capacity to express the phenotype of the desired tissue, and a biocompatible scaffold to deliver the cells to the damaged region. A literature review was performed using PubMed; peer-reviewed publications were screened for relevance in order to identify key advances in stem and progenitor cell contribution to the field of bone tissue engineering. In this review, we briefly introduce various adult stem cells implemented in bone tissue engineering such as mesenchymal stem cells (including bone marrow- and adipose-derived stem cells), endothelial progenitor cells, and induced pluripotent stem cells. We then discuss numerous advances associated with their application and subsequently focus on technological advances in the field, before addressing key regenerative strategies currently used in clinical practice. Stem and progenitor cell implementation in bone tissue engineering strategies have the ability to make a major impact on regenerative medicine and reduce patient morbidity. As the field of regenerative medicine endeavors to harness the body's own cells for treatment, scientific innovation has led to great advances in stem cell-based therapies in the past decade.

  17. Osteogenic cell lineage analysis is facilitated by organ cultures of embryonic chick periosteum.

    PubMed

    Bruder, S P; Caplan, A I

    1990-10-01

    Monoclonal antibodies against the surface of embryonic osteogenic cells (SB-1, SB-2, SB-3, and SB-5) have been used to characterize the sequence of transitions involved in the osteogenic cell lineage. In the present study, immunohistochemical analyses of the expression of osteogenic cell surface antigens in organ cultures of folded chick periosteum were performed. Unlike traditional culture methods using isolated osteoblastic cells, periosteal explants form a mineralized bone tissue in 4 to 6 days which is virtually identical to the in vivo counterpart. Examination of fresh explants confirm that no mature osteoblastic cells were present, although a discontinuous layer of preosteoblasts was evident. As the wave of osteodifferentiation swept through the cultured tissue, antibody SB-1 reacted with the surface of a large family of cells associated with the developing bone. Antibodies SB-3 and SB-2 reacted with progressively smaller subsets of cells, namely those in successively closer physical association with the newly formed and mineralizing bone. Cells recently encased in bone matrix were stained by both SB-2 and SB-5 antibodies, while those cells deep within the matrix reacted only with antibody SB-5. Analysis of this culture model allows dissection of the discrete cellular transition steps of osteogenesis, and reveals that osteogenic precursor cells proceed through the unique lineage stages which have been documented for in vivo osteogenesis. This culture system has furthermore provided evidence which is used to refine our understanding of the osteogenic cell lineage.

  18. Bone marrow mesenchymal stem cells stimulate cardiac stem cell proliferation and differentiation

    PubMed Central

    Hatzistergos, Konstantions E.; Quevedo, Henry; Oskouei, Behzad N.; Hu, Qinghua; Feigenbaum, Gary S.; Margitich, Irene S.; Mazhari, Ramesh; Boyle, Andrew J.; Zambrano, Juan P.; Rodriguez, Jose E.; Dulce, Raul; Pattany, Pradip M.; Valdes, David; Revilla, Concepcion; Heldman, A.W.; McNiece, I.; Hare, Joshua M.

    2012-01-01

    Rationale The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow derived mesenchymal stem cells (MSC) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes. Objective Here we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire. Methods And Results Female Yorkshire pigs (n=31) underwent experimental myocardial Infarction (MI); and 3 days later received transendocardial injections of allogeneic male bone marrow-derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit+ CSCs increased 20-fold in MSC treated animals vs. controls (p<0.001), there was a 6-fold increase in GATA-4+ CSCs in MSC vs. control (p<0.001), and mitotic myocytes increased 4-fold. Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit+ CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2-5 and troponin I. Conclusions MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics. PMID:20671238

  19. Autologous bone marrow stromal cells are promising candidates for cell therapy approaches to treat bone degeneration in sickle cell disease.

    PubMed

    Lebouvier, Angélique; Poignard, Alexandre; Coquelin-Salsac, Laura; Léotot, Julie; Homma, Yasuhiro; Jullien, Nicolas; Bierling, Philippe; Galactéros, Frédéric; Hernigou, Philippe; Chevallier, Nathalie; Rouard, Hélène

    2015-11-01

    Osteonecrosis of the femoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BM stromal cells. For this reason, expanded bone marrow stromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) compared with control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs from SCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products. PMID:26492634

  20. Autologous bone marrow stromal cells are promising candidates for cell therapy approaches to treat bone degeneration in sickle cell disease.

    PubMed

    Lebouvier, Angélique; Poignard, Alexandre; Coquelin-Salsac, Laura; Léotot, Julie; Homma, Yasuhiro; Jullien, Nicolas; Bierling, Philippe; Galactéros, Frédéric; Hernigou, Philippe; Chevallier, Nathalie; Rouard, Hélène

    2015-11-01

    Osteonecrosis of the femoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BM stromal cells. For this reason, expanded bone marrow stromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) compared with control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs from SCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products.

  1. Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones.

    PubMed

    Chagin, Andrei S; Karimian, Elham; Zaman, Farasat; Takigawa, Masaharu; Chrysis, Dionisios; Sävendahl, Lars

    2007-05-01

    Estrogen affects skeletal growth and promotes growth plate fusion in humans. High doses of estrogen have been used to limit growth in girls with predicted extreme tall stature; a treatment which has been associated with severe side effects. Selective estrogen receptor modulators (SERMs) could potentially be used as an alternative treatment. We chose to study the effects of Tamoxifen (Tam), a first generation SERM that has been used in the treatment of pubertal gynecomastia or McCune-Albright syndrome. Cultured fetal rat metatarsal bones were used to study the effects of Tam on longitudinal bone growth. In sectioned bones, chondrocyte apoptosis and proliferation were analyzed by TUNEL assay and BrdU incorporation, respectively. We also used a human chondrocytic cell line, HSC-2/8, to study the effects of Tam on apoptosis (FACS analysis and Cell Death detection ELISA) and caspase activation (caspase substrate cleavage and Western immunoblotting). Tam caused a dose-dependent growth retardation of cultured metatarsal bones. No catch-up growth was observed after Tam was removed from the culture medium. Detailed analysis of sectioned growth plate cartilage revealed increased apoptosis of chondrocytes within the resting and hypertrophic zones. HCS-2/8 cells also underwent apoptosis upon Tam treatment. Tam-induced apoptosis was caspase-dependent and completely abrogated by either caspase-8 or -9 inhibitors. A substrate assay revealed that caspase-8 is first activated followed by caspase-9 and -3. Finally, FasL secretion was stimulated by Tam and blocking of either FasL or Fas decreased Tam-induced apoptosis in chondrocytes. We here describe a novel mechanism of tamoxifen-induced apoptosis in chondrocytes, involving the activation of caspases and the FasL/Fas pathway, which diminishes the potential for bone growth.

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

    PubMed Central

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

    2014-01-01

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

  3. Human Perivascular Stem Cell-Based Bone Graft Substitute Induces Rat Spinal Fusion

    PubMed Central

    Chung, Choon G.; James, Aaron W.; Asatrian, Greg; Chang, Le; Nguyen, Alan; Le, Khoi; Bayani, Georgina; Lee, Robert; Stoker, David; Zhang, Xinli

    2014-01-01

    Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair. PMID:25154782

  4. The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

    PubMed

    Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

    2013-03-01

    Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

  5. IFITM1 increases osteogenesis through Runx2 in human alveolar-derived bone marrow stromal cells.

    PubMed

    Kim, Beom-Su; Kim, Hyung-Jin; Kim, Jin Seong; You, Yong-Ouk; Zadeh, Homa; Shin, Hong-In; Lee, Seung-Jin; Park, Yoon-Jeong; Takata, Takashi; Pi, Sung-Hee; Lee, Jun; You, Hyung-Keun

    2012-09-01

    The exact molecular mechanisms governing the differentiation of bone marrow stromal stem/progenitor cells (BMSCs) into osteoblasts remain largely unknown. In this study, a highly expressed protein that had a high degree of homology with interferon-induced transmembrane protein 1 (IFITM1) was identified using differentially expressed gene (DEG) screening. We sought to determine whether IFITM1 influenced osteoblast differentiation. During differentiation, IFITM1 expression gradually increased from 5 to 10days and subsequently decreased at 15 days in culture. Analysis of IFITM1 protein expression in several cell lines as well as in situ studies on human tissues revealed its selective expression in bone cells and human bone. Proliferation of human alveolar-derived bone marrow stromal cells (hAD-BMSCs) was significantly inhibited by IFITM1 knockdown by using short hairpin RNA, as were bone specific markers such as alkaline phosphatase, collagen type I α 1, bone sialoprotein, osteocalcin, and osterix were decreased. Calcium accumulation also decreased following IFITM1 knockdown. Moreover, IFITM1 knockdown in hAD-BMSCs was associated with inhibition of Runx2 mRNA and protein expression. Collectively, the present data provide evidence for the role of IFITM1 in osteoblast differentiation. The exact mechanisms of IFITM1's involvement in osteoblast differentiation are still under investigation.

  6. Three-dimensional polycaprolactone-hydroxyapatite scaffolds combined with bone marrow cells for cartilage tissue engineering.

    PubMed

    Wei, Bo; Yao, Qingqiang; Guo, Yang; Mao, Fengyong; Liu, Shuai; Xu, Yan; Wang, Liming

    2015-08-01

    The goal of this study was to investigate the chondrogenic potential of three-dimensional polycaprolactone-hydroxyapatite (PCL-HA) scaffolds loaded with bone marrow cells in vitro and the effect of PCL-HA scaffolds on osteochondral repair in vivo. Here, bone marrow was added to the prepared PCL-HA scaffolds and cultured in chondrogenic medium for 10 weeks. Osteochondral defects were created in the trochlear groove of 29 knees in 17 New Zealand white rabbits, which were then divided into four groups that underwent: implantation of PCL-HA scaffolds (left knee, n = 17; Group 1), microfracture (right knee, n = 6; Group 2), autologous osteochondral transplantation (right knee, n = 6; Group 3), and no treatment (right knee, n = 5; Control). Extracellular matrix produced by bone marrow cells covered the surface and filled the pores of PCL-HA scaffolds after 10 weeks in culture. Moreover, many cell-laden cartilage lacunae were observed, and cartilage matrix was concentrated in the PCL-HA scaffolds. After a 12-week repair period, Group 1 showed excellent vertical and lateral integration with host bone, but incomplete cartilage regeneration and matrix accumulation. An uneven surface of regenerated cartilage and reduced distribution of cartilage matrix were observed in Group 2. In addition, abnormal bone growth and unstable integration between repaired and host tissues were detected. For Group 3, the integration between transplanted and host cartilage was interrupted. Our findings indicate that the PCL-HA scaffolds loaded with bone marrow cells improved chondrogenesis in vitro and implantation of PCL-HA scaffolds for osteochondral repairenhanced integration with host bone. However, cartilage regeneration remained unsatisfactory. The addition of trophic factors or the use of precultured cell-PCL-HA constructs for accelerated osteochondral repair requires further investigation.

  7. Prognostic value of in vitro bone marrow culture in refractory anaemia with excess of myeloblasts.

    PubMed

    Faille, A; Dresch, C; Poirier, O; Balitrand, N; Najean, Y

    1978-03-01

    Bone marrow from 17 patients with refractory anaemia with excess of myeloblasts (RAEM) was cultured in methylcellulose semi-solid medium. Compared with normal bone marrow, 3 patterns of growth occurred corresponding with different clinical stages of the condition. Patients whose bone marrow grew normal colonies and those who produced a predominance of microclusters had the longest life expectance, while those who produced a predominance of macroclusters had the shortest life expectancy with a high rate of acute leukaemic transformation. Colony culture appears to be a useful prognostic tool in the condition.

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

    SciTech Connect