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Sample records for bone cell cultures

  1. 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 methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

  2. 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 same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.

  3. Spaceflight effects on cultured embryonic chick bone cells.

    PubMed

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

    2000-06-01

    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 same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general. PMID:10841178

  4. 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.

  5. 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-Thrse; 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

  6. Benefits of hypoxic culture on bone marrow multipotent stromal cells

    PubMed Central

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

    2012-01-01

    Cultivation of cells is usually performed under atmospheric oxygen tension; however, such a condition does not replicate the hypoxic conditions of normal physiological or pathological status in the body. Recently, the effects of hypoxia on bone marrow multipotent stromal cells (MSCs) have been investigated. In a long-term culture, hypoxia can inhibit senescence, increase the proliferation rate and enhance differentiation potential along the different mesenchymal lineages. Hypoxia also modulates the paracrine effects of MSCs, causing upregulation of various secretable factors, including the vascular endothelial growth factor and interleukin-6, and thereby promoting wound healing and diabetic fracture healing. Finally, hypoxia plays an important role in mobilization and homing of MSCs, primarily by its ability to induce stromal cell-derived factor-1 expression along with its receptor, CXCR4. After transplantation, an ischemic environment, that is the combination of hypoxia and lack of nutrition, can lead to apoptosis or cell death, which can be overcome by the hypoxic preconditioning of MSCs and overexpression of prosurvival genes like Akt, HO-1 and Hsp70. This review emphasizes that hypoxia is an important factor in all major aspects of stem cell biology, and the mechanism involved in the hypoxic inducible factor-1signaling pathway behind these responses is also discussed. PMID:23119226

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

    PubMed Central

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

    2010-01-01

    We report engineering of half-centimeter–sized bone constructs created in vitro using human adipose-derived stem cells (hASCs), decellularized bone scaffolds, and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4 mm diameter × 4 mm thick), providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400 μm/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-β-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture, and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs. PMID:19678762

  8. 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

  9. Characterization of natural killer cells cultured from human bone marrow cells

    SciTech Connect

    Yoda, Y.; Kawakami, Z.; Shibuya, A.; Abe, T.

    1988-09-01

    Human bone marrow (BM) cells, depleted of nylon wool-adherent cells, T cells, and natural killer (NK) cells, were cultured in medium containing recombinant interleukin 2 (rIL2). After 21 or 24 days in culture, numerous lymphoid cells with multiple azurophilic granules and a morphology similar to large granular lymphocytes (LGL) were found. Two-color analysis of surface phenotype showed many of these cells to be NKH1-positive and a limited number of cells had other NK markers such as CD16, CD2, or CD8. The CD3 antigen was not coexpressed with NKH1. The cultured BM cells were cytotoxic for K562, Daudi, and Raji cell lines. The NKH1+, CD2-, CD3-, CD16- cells were sorted and, in addition to having the LGL morphology, were found to be cytotoxic for K562 cells (NK (K562)). The generation of NK(K562) activity was significantly suppressed by 5-bromodeoxyuridine plus ultraviolet light treatment, indicating that DNA synthesis is required. These experiments suggest that the described culture conditions allow differentiation of progenitor cells, into immature, but functionally active, NK cells.

  10. 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

  11. Guided differentiation of bone marrow stromal cells on co-cultured cartilage and bone scaffolds.

    PubMed

    Lee, Paul; Tran, Katelyn; Zhou, Gan; Bedi, Asheesh; Shelke, Namdev B; Yu, Xiaojun; Kumbar, Sangamesh G

    2015-10-14

    Focal chondral defects that result from traumatic injuries to the knee remain one of the most common causes of disability in patients. Current solutions for healing focal cartilage defects are mainly limited by the production of inferior cartilage-like tissue and subsequent delamination due to incomplete healing of the subchondral bone. In this experiment a polymeric osteochondral implant for guiding autologous bone marrow stem cells (BMSCs) to populate the scaffold to create distinctive bone and cartilage tissue is used. The cartilage component presents bioactive aligned nanofibers containing chondroitin sulfate and hyaluronic acid while the bone component includes hydroxyapatite to promote chondrogenic and osteogenic differentiation of the rat BMSCs in vitro. The different cartilage and bone components resulted in the elevated expression of osteogenic markers such as bone sialoprotein, runt related transcription factor 2, and bone morphogenetic protein 2 in the deeper bone layer and chondrogenic markers such as collagen type II and aggrecan in the cartilage layer. Through immunofluorescence imaging, the alignment of the secreted collagen type II fibrils and aggrecan was visualized and quantified on the cartilage component of the scaffold. These current studies show that the biodegradable biphasic osteochondral implant may be effective in promoting more hyaline-like tissue to fill in chondral defects of the knee. PMID:26292727

  12. Osteoblastic cells regulate mi gene expression in cultured bone marrow cells.

    PubMed

    Kawaguchi, N; Noda, M

    1998-04-01

    The microphthalmia (mi) gene encodes a bHLH-Zip protein and certain mutations in this gene are known to result in osteopetrosis. We examined mi gene expression in murine bone marrow (BM) cells and calvaria-derived osteoblastic (OB) cells. The mi message level in BM cells was three- to four-fold higher than that in OB cells. The mi message level in BM cells alone was not enhanced by 1,25(OH)2 vitamin D3 (vitamin D) which induces TRAP-positive multinucleated cells, but was enhanced two-fold by the addition of conditioned medium obtained from cultures of OB cells. The mi message levels in BM cells were also enhanced by macrophage colony-stimulating factor (M-CSF). The mi message levels in cocultures of BM cells and osteoblastic MC3T3-E1 cells, which do not support osteoclastogenesis, were similar to those in the cocultures of BM cells and OB cells. Cocultures of BM cells with MC3T3-E1 cells did not yield TRAP-positive multinucleated cells, but they did maintain osteoclast progenitor cells. These findings suggest that mi is expressed in BM cells that may be under the control of OB cells. PMID:9650449

  13. [Biocompatibility of a new ionomer bone cement in endoprosthetics. In vitro testing using a mixed bone cell culture].

    PubMed

    Meyer, U; Szulczewski, D H; Möller, K; Jones, D B; Wuisman, P

    1996-01-01

    Periosteal derived bovine osteoblasts and osteoclasts migrated in culture onto an ionomeric cement, currently evaluated as a cement for orthopaedic implants. Cell cultures were maintained for 4 weeks and used to study the in-vitro behaviour of cells on the material surface. Osteoblasts and osteoclasts colonized the substrate in monolayers and exhibited their phenotypic morphology. Differentiation into both cell lines were demonstrated by immunostaining. Staining for aluminium in cells growing on the bone cement showed an uptake and storage of aluminium in the cells. EDAX-microanalysis revealed high concentrations of Al and Si in the periosteal tissue. Despite uptake of Al in the cells, signs of toxicity were not apparent in the cell culture system. PMID:8779254

  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. Osteogenic performance of donor-matched human adipose and bone marrow mesenchymal cells under dynamic culture.

    PubMed

    Wu, Wei; Le, Andrew V; Mendez, Julio J; Chang, Julie; Niklason, Laura E; Steinbacher, Derek M

    2015-05-01

    Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7-21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells. PMID:25668104

  16. Calcitonin receptors as markers for osteoclastic differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures.

    PubMed

    Hattersley, G; Chambers, T J

    1989-09-01

    The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, but analysis of lineage and regulation of differentiation has been hampered by lack of a specific marker that enables identification of cells of osteoclastic phenotype. Previously used markers, such as multinuclearity, that are specific for osteoclasts in bone become less specific in culture. Uniquely among bone and bone marrow cells, osteoclasts possess abundant calcitonin (CT) receptors. We therefore tested the correlation between the generation of bone-resorptive function and the formation of CT receptor-positive cells from hemopoietic tissue in vitro. Without 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], a hormone that induces osteoclastic differentiation in vitro, bone marrow cultures showed very little bone resorption, and only small numbers of CT receptor-positive cells developed. When 1,25-(OH)2D3 was added to the cultures, CT receptor-positive cells developed within 1 day and reached a peak after 7 days. Bone resorption commenced within 2 days of hormone addition. There was a strong parallelism between the cumulative number of CT receptor-positive cells and the extent of bone resorption. The capacity of cultures to generate bone-resorptive activity and CT receptor-positive cells declined progressively when 1,25-(OH)2D3 was added to hemopoietic tissue after a 7- to 21-day hormone-free incubation period. The number of CT receptor-positive cells in these cultures correlated strongly (r = 0.96) with bone resorption. The behavior of these cultures suggests that 1,25-(OH)2D3 acts to induce terminal differentiation of osteoclast precursors present in the cultures, and that precursor cell numbers decreased with increasing time in vitro. All of the CT receptor-positive cells in control cultures and all of those seen shortly after 1,25-(OH)2D3 addition were mononuclear, despite considerable bone resorption; the majority of CT receptor-positive cells remained mononuclear throughout the incubation period. This suggests that mononuclear cells with characteristics of osteoclasts exist that are able to excavate bone. CT receptor-positive cells slightly preceded the development of bone-resorptive function, implying that CT receptors develop before the acquisition of bone-resorptive capacity by osteoclasts. Peritoneal macrophages, blood mononuclear cells, and cells of the J774 macrophage cell line failed to either resorb bone or express CT receptors, even after incubation with 1,25-(OH)2D3 for 14 days. These results show a strong and specific correlation between the generation of bone-resorptive cells and CT receptor-positive cells, and suggest that CT receptor express PMID:2547591

  17. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1983-07-01

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

  19. Evaluation ofprimary blood monocyte and bone marrow cell culture for the isolation of Rickettsia rickettsii.

    PubMed

    Buhles, W C; Huxsoll, D L; Ruch, G; Kenyon, R H; Elisberg, B L

    1975-12-01

    Rickettsia rickettsii was isolated from experimentally infected guinea pigs by culture of blood monocytes and bone marrow cells, and from experimentally infected rhesus monkeys by blood monocyte culture. Rickettsiae were identified in monocyte-macrophage monolayers stained by Gimnez or flourescent antibody techniques. A total of 78 culture attempts were made from 20 guinea pigs and 16 monkeys. The success of isolation of R. rickettsii in culture was positively correlated with the numbers of rickettsiae present in the blood and bone marrow. in cultures derived from infected guinea pigs, rickettsiae were usually observed after 5 to 7 days of culture, and in monkeys monocyte cultures they were usually observed within 3 to 5 days. Positive cultures were derived from guinea pigs and monkeys as early as the first day of fever and 1 to 3 days before the appearance of other clinical signs. Monocyte cultures became negative with the resolution of rickettsemia and concomitantly with the appearance of serum antibody. Monocyte culture isolation of R. rickettsii may be as sensitive for the detection of rickettsiae in blood and marrow as the intraperitoneal inoculation of guinea pigs or the plaque assay technique. Because of the simplicity of the method and because rickettsiae were often identified within 3 to 5 days after initiation, the monocyte culture technique may be useful in the early diagnois of human rickettsial disease. PMID:812828

  20. 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 bioluminescence imaging (BLI). Although the cultured MSCs had osteogenic potential, their contribution to spinal fusion when seeded in mineral scaffolds, in the conditions disclosed here, remains uncertain probably due to callus interference with the scaffolds. At present, bone autografts are better than hybrid constructs for posterolateral lumbar fusion, but we should continue to seek better conditions for efficient tissue engineering. PMID:25522168

  1. Dynamic cell culture on calcium phosphate microcarriers for bone tissue engineering applications

    PubMed Central

    Perez, Roman A; Riccardi, Kiara; Altankov, George

    2014-01-01

    Developing appropriate cell culturing techniques to populate scaffolds has become a great challenge in tissue engineering. This work describes the use of spinner flask dynamic cell cultures to populate hydroxyapatite microcarriers for bone tissue engineering. The microcarriers were obtained through the emulsion of a self-setting aqueous α-tricalcium phosphate slurry in oil. After setting, hydroxyapatite microcarriers were obtained. The incorporation of gelatin in the liquid phase of the α-tricalcium phosphate slurry allowed obtaining hybrid gelatin/hydroxyapatite-microcarriers. Initial cell attachment on the microcarriers was strongly influenced by the speed of the dynamic culture, achieving higher attachment at low speed (40 r/min) as compared to high speed (80 r/min). Under moderate culture speeds (40 r/min), the number of cells present in the culture as well as the number of microcarrier-containing cells considerably increased after 3 days, particularly in the gelatin-containing microcarriers. At longer culture times in dynamic culture, hydroxyapatite-containing microcarriers formed aggregates containing viable and extracellular matrix proteins, with a significantly higher number of cells compared to static cultures. PMID:25383168

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

    PubMed

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

    2013-01-01

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

  3. High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture.

    PubMed

    Takahashi, Etsuko; Mukohyama, Hitoshi; Aoki, Kazuhiro; Duarte, Wagner R; Lerner, Ulf H; Ohya, Keiichi; Omura, Ken; Kasugai, Shohei

    2002-12-01

    The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system. PMID:12641381

  4. Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system.

    PubMed

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

    2015-10-01

    In the bone marrow, hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment. Previously, we established a 3D bone marrow culture system. In this study, we analyzed the kinetics of hematopoietic cells, and more than 50% of hematopoietic progenitor cells, including CFU-Mix, CFU-GM and BFU-E in 3D culture were in a resting (non-S) phase. Furthermore, we examined the hematopoietic supportive ability of stromal cells by measuring the expression of various mRNAs relevant to hematopoietic regulation. Over the 4 weeks of culture, the stromal cells in the 3D culture are not needlessly activated and "quietly" regulate hematopoietic cell proliferation and differentiation during the culture, resulting in the presence of resting hematopoietic stem cells in the 3D culture for a long time. Thus, the 3D culture system may be a new tool for investigating hematopoietic stem cell-stromal cell interactions in vitro. PMID:26431462

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

    PubMed

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

    2011-11-01

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

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

    PubMed Central

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

    2011-01-01

    Purpose The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2. Methods MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days. Results At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7. Conclusions The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium. PMID:22087413

  8. 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-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions. PMID:25469312

  9. Genetic control of mast cell development in bone marrow cultures. Strain-dependent variation in cultures from inbred mice.

    PubMed Central

    Reed, N D; Wakelin, D; Lammas, D A; Grencis, R K

    1988-01-01

    A comparison was made of the capacity of bone marrow cells (BM) from genetically distinct strains of mice to develop into mast cells under defined conditions of in vitro culture. In the presence of conditioned media derived from ConA treated spleen cells from normal or Trichinella spiralis-infected mice, mast cell development occurred readily. After 21 days of culture mast cells comprised more than 90% of the total cell population. BM taken from certain strains of mice (SWR and NIH) produced large numbers of mast cells, total cell numbers increasing between 2 and 5 fold; other strains (C57BL/10 [B10] B10 congenics) produced relatively few mast cells, total cell numbers not increasing above the starting concentration or declining during culture. The genetic factors determining the strain-response phenotype (no. of mast cells in culture) were predominantly associated with the background genome. No significant differences in response were noted between the B10 congenic strains B10 [H-2b], B10.G [H-2q] or B10.BR [H-2k], which differ only at the MHC, whereas major differences were seen between B10.G and the other H-2q strains [SWR and NIH]. Response phenotype was not inherited as a simple dominant trait; F1 progeny of high x low responder strains were intermediate between the parental values. The expression of genetic influences upon mast cell response phenotype appears to be at both the level of mast cell precursor cells, as determined from limiting dilution assays of BM from high, low and F1 (high x low) strains, and at the level of mast cell proliferation, as determined by repeated sub-culture of mast cells from these strains. PMID:3208455

  10. 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

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

    PubMed

    Curtin, Paul; Youm, Helen; Salih, Erdjan

    2012-02-01

    One of the major limitations of studying cancer-bone metastasis has been the lack of an appropriate ex-vivo model which can be used under defined conditions that simulates closely the in vivo live bone microenvironment in response to cancer-bone interactions. We have developed and utilized a three-dimensional (3D) cancer-bone metastasis model using free-floating live mouse calvarial bone organs in the presence of cancer cells in a roller tube system. In such co-cultures under hypoxia and a specifically defined bone remodeling stage, viz., resorption system, cancer cells showed a remarkable affinity and specificity for the "endosteal side" of the bone where they colonize and proliferate. This was concurrent with differentiation of resident stem/progenitor cells to osteoclasts and bone resorption. In contrast, under bone formation conditions this model revealed different pathophysiology where the breast cancer cells continued to induce osteoclastic bone resorption whereas prostate cancer cells led to osteoblastic bone formation. The current 3D model was used to demonstrate its application to studies involving chemical and biochemical perturbations in the absence and presence of cancer cells and cellular responses. We describe proof-of-principle with examples of the broad versatility and multi-faceted application of this model that adds another dimension to the ongoing studies in the cancer-bone metastasis arena. PMID:22071100

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

    SciTech Connect

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

    1986-07-15

    Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period.

  13. Pasteurella multocida toxin is a mitogen for bone cells in primary culture.

    PubMed Central

    Mullan, P B; Lax, A J

    1996-01-01

    The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage. PMID:8641807

  14. 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. PMID:22473741

  15. 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

  16. Effect of the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells in vitro.

    PubMed

    Lin, Qiwang; Wang, Linhui; Bai, Yuling; Hu, Menglin; Mo, Jianling; He, Haixin; Lou, Aiju; Yang, Bo; Zhao, Hongpu; Guo, Yuan; Wu, Yanfeng; Wang, Le

    2016-06-01

    Mesenchymal stem cells (MSCs) give origin to the marrow tromal environment that supports hematopoiesis. These cells present a wide range of differentiation potentials and a complex relationship with hematopoietic stem cells (HSCs) and endothelial cells. In addition to bone marrow (BM), MSCs can be obtained from other sites in the adult or the fetus. Recent studies have shown that cocultured endothelial cells and osteoblasts are mutually promotive in bone tissues repair. In this study, we observed the effects of coculture of endothelial cells and osteoblasts at different ratios on vasculogenesis and bone formation, and we found that angiogenic effect is more effective when endothelial cells are cocultured with osteoblasts at the ratio of 4:1, and osteogenic effect is more effective at the ratio of 1:4. It is concluded that the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells could be a promising culture system for bone tissue engineering applications. PMID:26479150

  17. 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. PMID:25335987

  18. The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles

    PubMed Central

    Bentivegna, Angela; Roversi, Gaia; Riva, Gabriele; Paoletta, Laura; Redaelli, Serena; Miloso, Mariarosaria; Tredici, Giovanni; Dalprà, Leda

    2016-01-01

    Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures. PMID:26880970

  19. The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles.

    PubMed

    Bentivegna, Angela; Roversi, Gaia; Riva, Gabriele; Paoletta, Laura; Redaelli, Serena; Miloso, Mariarosaria; Tredici, Giovanni; Dalprà, Leda

    2016-01-01

    Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures. PMID:26880970

  20. Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells.

    PubMed

    Vidyasekar, Prasanna; Shyamsunder, Pavithra; Sahoo, Sanjeeb Kumar; Verma, Rama Shanker

    2016-02-01

    3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived. PMID:26542170

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. Hyaluronan Binding Identifies a Functionally Distinct Alveolar Macrophage-like Population in Bone Marrow-Derived Dendritic Cell Cultures.

    PubMed

    Poon, Grace F T; Dong, Yifei; Marshall, Kelsey C; Arif, Arif; Deeg, Christoph M; Dosanjh, Manisha; Johnson, Pauline

    2015-07-15

    Although classical dendritic cells (DCs) arise from distinct progenitors in the bone marrow, the origin of inflammatory DCs and the distinction between monocyte-derived DCs and macrophages is less clear. In vitro culture of mouse bone marrow cells with GM-CSF is a well-established method to generate DCs, but GM-CSF has also been used to generate bone marrow-derived macrophages. In this article, we identify a distinct subpopulation of cells within the GM-CSF bone marrow-derived DC culture based on their ability to bind hyaluronan (HA), a major component of the extracellular matrix and ligand for CD44. HA identified a morphologically distinct subpopulation of cells within the immature DC population (CD11c(+) MHC II(mid/low)) that were CCR5(+)/CCR7(-) and proliferated in response to GM-CSF, but, unlike immature DCs, did not develop into mature DCs expressing CCR7 and high levels of MHC II, even after stimulation with LPS. The majority of these cells produced TNF-α in response to LPS but were unable to activate naive T cells, whereas the majority of mature DCs produced IL-12 and activated naive T cells. This HA binding population shared many characteristics with alveolar macrophages and was retained in the alveolar space after lung instillation even after LPS stimulation, whereas the MHC II(high) mature DCs were found in the draining lymph node. Thus, HA binding in combination with MHC II expression can be used to identify alveolar-like macrophages from GM-CSF-treated bone marrow cultures, which provides a useful in vitro model to study alveolar macrophages. PMID:26085682

  8. 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. PMID:26999801

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

    PubMed

    Grssel, Susanne; Stckl, Sabine; Jenei-Lanzl, Zsuzsa

    2012-01-01

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

  10. Ki-energy (Life-energy) Stimulates Osteoblastic Cells and Inhibits the Formation of Osteoclast-like Cells in Bone Cell Culture Models

    PubMed Central

    Nishino, Kozo; Uchiyama, Satoshi; Ohnishi, Tomoko; Yamaguchi, Masayoshi

    2007-01-01

    Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10 min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72 h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P < 0.01 with n = 8). We performed a reverse transcription-polymerase chain reaction (RT–PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. After 48 h, RT–PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P < 0.05 and n = 4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1–34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10 min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P < 0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored. PMID:17549240

  11. 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.

  12. 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 mechanosensitivity of bone cells is altered under near weightlessness conditions, and that this abnormal mechanosensation contributes to disturbed bone metabolism observed in astronauts. In our current project for the International Space Station, we wish to test this hypothesis experimentally using an in vitro model. The specific aim of our research project is to test whether near weightlessness decreases the sensitivity of bone cells for mechanical stress through a decrease in early signaling molecules (NO, PGs) that are involved in the mechanical loading-induced osteogenic response. Bone cells are cultured with or without gravity prior to and during mechanical loading, using our modified in vitro oscillating fluid flow apparatus. In this "FlowSpace" project we are developing a cell culture module that is used to provide further insight in the mechanism of mechanotransduction in bone. PMID:15000126

  13. 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 mechanosensitivity of bone cells is altered under near weightlessness conditions, and that this abnormal mechanosensation contributes to disturbed bone metabolism observed in astronauts. In our current project for the International Space Station, we wish to test this hypothesis experimentally using an in vitro model. The specific aim of our research project is to test whether near weightlessness decreases the sensitivity of bone cells for mechanical stress through a decrease in early signaling molecules (NO, PGs) that are involved in the mechanical loading-induced osteogenic response. Bone cells are cultured with or without gravity prior to and during mechanical loading, using our modified in vitro oscillating fluid flow apparatus. In this "FlowSpace" project we are developing a cell culture module that is used to provide further insight in the mechanism of mechanotransduction in bone.

  14. Human bone marrow and umbilical cord blood cells generate CD4+ and CD8+ single-positive T cells in murine fetal thymus organ culture.

    PubMed Central

    Yeoman, H; Gress, R E; Bare, C V; Leary, A G; Boyse, E A; Bard, J; Shultz, L D; Harris, D T; DeLuca, D

    1993-01-01

    Murine fetal thymus lobes isolated from both normal and scid/scid mice can be colonized by donor cells from either human bone marrow or human umbilical cord blood in vitro. Subsequent organ culture results in a transient production of a few CD4+ CD8+ (double-positive) cells and then the accumulation of CD4+ or CD8+ (single-positive) T cells. A significant number of immature T-cell intermediates (e.g., CD8low, CD3-/low cells) were present in early organ cultures, suggesting that these were progenitors of the mature CD3+/high single-positive T cells that dominated late cultures. Depletion of mature T cells from the donor-cell populations did not affect their ability to colonize thymus lobes. However, colonization depended on the presence of CD7+ progenitor T cells. Limiting dilution experiments using mature T-cell populations (human peripheral blood leukocytes, human bone marrow cells, and human umbilical cord blood cells) suggested that thymic organ culture supports the growth of progenitor T cells but does not support the growth of mature human T cells. Each of these donor populations produced single-positive populations with different CD4/CD8 ratios, suggesting that precursor cells from different sources differ qualitatively in their capacity to differentiate into T cells. Images Fig. 1 PMID:7902570

  15. The role of LFA-1 in osteoclast development induced by co-cultures of mouse bone marrow cells and MC3T3-G2/PA6 cells.

    PubMed

    Tani-Ishii, N; Penninger, J M; Matsumoto, G; Teranaka, T; Umemoto, T

    2002-06-01

    Interactions between leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) influence the development of osteoclasts. However, little is known about how these adhesion molecules are involved in the process of osteoclast development. This study evaluated the role of LFA-1 and its ligands in osteoclast development and bone resorption. Co-cultures of bone marrow cells from LFA-1-deficient mice and MC3T3-G2/PA6 (PA6) cells were cultured in the presence of 1alpha,25(OH)2D3 and dexamethasone for 7 days. The number of TRAP-positive cells that were generated by bone marrow cells from LFA-1-deficient mice was smaller than that generated by bone marrow cells from wild-type mice. In addition, the bone-resorbing activity of osteoclast-like cells that were generated from LFA-1-deficient mice was lower than that generated by osteoclast-like cells from wild-type mice. Immunofluorescence flow cytometry showed that osteoclast stromal PA6 cells expressed the cell adhesion molecules, ICAM-1 and VCAM-1. When monoclonal antibodies to mice VCAM-1, CD11b or CD18 were added separately to the co-culture system, the number of TRAP-positive cells that were generated from LFA-1-deficient mice was 20-30% smaller than that generated from wild-type mice. The formation of TRAP-positive cells from both LFA-1 deficient and wild-type mice was especially inhibited by anti-CD18 antibody, in comparison to the addition of normal IgG serum. These results suggest that LFA-1 adhesion molecules play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. CD18 appears to be a key adhesion molecule in cell-to-cell contacts during the early stage of osteoclast development. PMID:12113552

  16. Effect of culture substrates and fibroblast growth factor addition on the proliferation and differentiation of rat bone marrow stromal cells.

    PubMed

    Hori, Yukana; Inoue, Sachiko; Hirano, Yoshiaki; Tabata, Yasuhiko

    2004-01-01

    This study is an investigation of the proliferation and differentiation of bone marrow stromal cells (BMSCs) on film substrates with different surface properties. Films of noncharged polymers with several water wettabilities; cell culture plates coated with collagen type I or IV, gelatin, or basic fibroblast growth factor (bFGF); and glass were used. BMSCs isolated from rat bone marrow were cultured on the various substrates in medium with dexamethasone [Dex(+)] or without dexamethasone [Dex(-)] to assess cell proliferation and differentiation. The number of proliferated BMSCs depended on the water wettability of substrates, although the cell number was greater in Dex(-) medium than in Dex(+) medium. Protein-coated substrates exhibited a high proliferation rate compared with noncoated substrates. Alkaline phosphatase (ALP) activity increased with increasing cell number, whereas ALP activity per cell correlated well with cell number. When cultured in Dex(+) medium containing bFGF or on culture plates coated with bFGF, BMSC proliferation tended toward enhancement with an increase in the amount of bFGF added in solution form, whereas it did not depend on the amount of bFGF in coated form. On the other hand, ALP activity and calcium content of BMSCs became maximal with bFGF coated at about 1 x 10(3) to 2 x 10(3) ng, in contrast to bFGF in solution form. Irrespective of the amount of bFGF, ALP activity and calcium content levels for bFGF in coated form were higher than for bFGF in solution form. It is concluded that the type of culture substrate and the manner of addition of bFGF affect the proliferation and differentiation of BMSCs. PMID:15363157

  17. 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, and that this abnormal mechanosensation contributes to disturbed bone metabolism observed in astronauts.In our current project for the International Space Station, we wish to test this hypothesis experimentally using an in vitro model. The specific aim of our research project is to test whether near weightlessness decreases the sensitivity of bone cells for mechanical stress through a decrease in early signaling molecules (NO, PGs) that are involved in the mechanical loading-induced osteogenic response. Bone cells are cultured with or without gravity prior to and during mechanical loading, using our modified in vitro oscillating fluid flow apparatus. In this "FlowSpace" project we are developing a cell culture module that is used to provide further insight in the mechanism of mechanotransduction in bone.

  18. 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.; Dept. of Material Science and Metallurgical Engineering, Technical University of Catalonia , ETSEIB, Av. Diagonal 647, 08028 Barcelona; CIBER-BBN, Maria de Luna 11, Ed. CEEI, 50118 Zaragoza ; 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.

  19. Improved proteomic profiling of the cell surface of culture-expanded human bone marrow multipotent stromal cells.

    PubMed

    Mindaye, Samuel T; Ra, Moonjin; Lo Surdo, Jessica; Bauer, Steven R; Alterman, Michail A

    2013-01-14

    A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry. This report presents the first comprehensive proteomic analysis of membrane proteome of undifferentiated and culture-expanded human bone marrow multipotent stromal cells (hBM-MSC) obtained from different human donors. Gene ontology mapping using the Ingenuity Pathway Analysis and DAVID programs revealed the largest membrane proteomic dataset for hBM-MSC reported to date. Collectively, the new workflow enabled us to identify at least two-fold more membrane proteins compared to published results on hBM-MSC. A total of 84 CDs were identified including 14 CDs identified for the first time. This dataset can serve as a basis for further exploration of self-renewal, differentiation and characterization of hBM-MSC. PMID:23153793

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

    PubMed Central

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

    2014-01-01

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

  1. Defective adhesion and homing of hemopoietic precursors from the bone marrow of patients with myelodysplasia to stromal cells of normal bone marrow cultures.

    PubMed

    Manakova, T E; Tsvetaeva, N V; Belkin, V M; Momotyuk, K S; Khoroshko, N D

    2005-01-01

    Hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome were characterized by lower adhesion to normal stromal sublayer compared to bone marrow precursors from healthy donors, while adhesion to fibroblast monolayer and fibronectin was similar in bone marrow cells from patients and donors. In vitro experiments showed that the percentage of adherent hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome in normal stromal sublayer and fibroblasts was lower compared to healthy donors. The decrease in adhesive activity of hemopoietic precursors from the bone marrow of patients with myelodysplastic syndrome probably contributes to impairment of cell-cell interactions in the bone marrow of these patients. PMID:16142262

  2. 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.

  3. 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, erythroid, or other cell lines would be studied. Transfection with a known gene would be attempted and then conversion to a terminally identifiable cell.

  4. Culture of bone-marrow-derived cells in microfabricated pit arrays

    NASA Astrophysics Data System (ADS)

    Kikuchi, Hiroko E.; Kikuchi, Yuji; Kuboki, Yoshinori

    2001-05-01

    Tissue cells cannot survive without the attachment to extracellular matrix (ECM). Furthermore, the geometry of ECM is known to play an essential role in the regulation for these cells to proliferate and differentiate so that tissues with normal morphologies can be formed or maintained. We have introduced microfabricated surface structures coated with ECM protein collagen into cell culture studies to examine a possibility of 3D patterning of ECM.

  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. PMID:24859219

  6. Micro-PIXE and FT-IR microscopic studies of mineralized nodules formed in rat bone marrow stromal cell cultures

    NASA Astrophysics Data System (ADS)

    Rokita, E.; Mutsaers, P. H. A.; Quaedackers, J. A.; Tatoń , G.; de Voigt, M. J. A.

    1998-04-01

    A proton microprobe in combination with Proton Induced X-ray Emission (micro-PIXE) and Fourier transform-infrared (FT-IR) microscopy are used for examination of the elemental composition and structure of inorganic deposits formed in cell cultures of rat bone marrow. The results show that micro-PIXE is well suited for prior recognition of the localization and for the determination of the elemental composition of the deposits while FT-IR microscopy may be used to determine the structure of deposits in situ with a spatial resolution of about 20 μm. It is demonstrated that nodules in the cell culture are composed of different Ca compounds. In contrast to physiological mineralization, Ca-P compounds do not dominate the mineral phase. Discrepancies are observed between histomorphometric and micro-PIXE data.

  7. Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Su, Xiaohu; Ling, Yu; Liu, Chunxia; Meng, Fanhua; Cao, Junwei; Zhang, Li; Zhou, Huanmin; Liu, Zongzheng; Zhang, Yanru

    2015-08-01

    We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.70.05?h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research. PMID:26086202

  8. The Role of Glucose, Serum, and Three-Dimensional Cell Culture on the Metabolism of Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Deorosan, Byron; Nauman, Eric A.

    2011-01-01

    Mesenchymal stem cells (MSCs) have become a critical addition to all facets of tissue engineering. While most in vitro research has focused on their behavior in two-dimensional culture, relatively little is known about the cells' behavior in three-dimensional culture, especially with regard to their metabolic state. To evaluate MSC metabolism during twodimensional culture, murine bone marrow-derived MSCs were cultured for one week using twelve different medium compositions, varying in both glucose and fetal bovine serum (FBS) concentrations. The results indicate that glucose concentration was the more important factor in sustaining cell growth and viability. To evaluate metabolic state during three-dimensional culture, MSCs were cultured for one week using two different medium compositions and two different concentrations of collagen gel matrix. The medium compositions only varied in glucose concentration. The results indicate that glucose and extracellular matrix were significant factors in the metabolic response of the cells. However, cells cultured in low density collagen exhibited considerable cell death, likely because of physical contraction of the collagen hydrogel which was not observed in the higher density collagen. These findings will be useful to the development of in vitro cell culture models that properly mimic in vivo physiological processes. PMID:21603146

  9. Neurotrophic actions of bone marrow stromal cells on primary culture of dorsal root ganglion tissues and neurons.

    PubMed

    Gu, Yun; Wang, Jie; Ding, Fei; Hu, Nan; Wang, Yaxian; Gu, Xiaosong

    2010-03-01

    Application of adult bone marrow stromal cells (BMSCs) provides therapeutic benefits to the treatment of neurological insults. The aim of this study was to explore the potential of nonhematopoietic BMSCs to produce soluble factors and stimulate signaling pathways in neurons that mediate trophic effects. A combination of enzyme-linked immunosorbent assay and two-dimensional gel electrophoresis coupled with mass spectrometry showed that the BMSCs released into the culture medium an array of soluble factors such as nerve growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, and ciliary neurotrophic factor, which have been shown to exhibit potent neurotrophic effects on neural cells. Immunochemistry, cell viability assay, and quantitative real-time RT-PCR collectively showed that neurite outgrowth and neurogenesis in cultured rat dorsal root ganglion (DRG) explants and neurons were enhanced after they were cocultured with rat BMSCs. Western blot analysis revealed that BMSC-conditioned medium activated phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphoinositide 3-kinase/serine/threonine kinase (PI3K/Akt) in primary culture of rat DRG neurons, which suggested that BMSCs trigger endogenous survival signaling pathways in neurons through their secreted soluble factors. Our data help to elucidate the mechanisms by which BMSCs function as a cell therapy agent in peripheral nerve regeneration. PMID:19894026

  10. [Standardized testing of bone implant surfaces with an osteoblast cell culture system. II. Titanium surfaces of different degrees of roughness].

    PubMed

    Nöth, U; Hendrich, C; Merklein, F; Altvater, T; Rader, C P; Schütze, N; Eulert, J; Thull, R

    1999-01-01

    The effect of titanium surfaces with different degrees of roughness on osteoblast proliferation and differentiation was investigated using a standardised cell culture system. Human foetal osteoblasts (hFOB 1.19) were cultured on polished (Ti pol), sandblasted (Ti sb) and sandblasted/heat treated (Ti sb-ht) titanium surfaces for 17 days. Cell culture quality polystyrene (Ps) was used as a control. Cell number and viability were determined for assessment of proliferation. Alkaline phosphatase activity, collagen I and osteocalcin production were measured as parameters for osteoblast differentiation. In the early phase, higher proliferation values were measured on Ti pol. However, on Ti sb and Ti sb-ht higher proliferation was found in the late phase. The activity of the early differentiation marker alkaline phosphatase was higher on Ti pol. No differences were seen for the late differentiation parameters collagen I and osteocalcin. The test system permits the influence of the surface structure on the dynamics of the osteoblast development cycle to be determined. The larger surface area of rough materials leads to an initially delayed, but then prolonged cell proliferation. This model correlates with recent in vivo findings, and confirms the use of rough surfaces for implants in direct contact with bone, even at the cellular level. PMID:10194879

  11. 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.

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

    PubMed

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

    2013-09-11

    Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell-culture methods are ill-suited to study the intricacies of bone biology because they ignore the three-dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2-3 mm thickness. In this project, a flow-perfusion system was custom-designed to overcome these limitations: eight adaptable chambers housed cylindrical cell-seeded scaffolds measuring 12 or 24 mm in diameter and 1-10 mm in thickness. The porous scaffolds were manufactured using a three-dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi-channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas-permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor-scaffold system, using 12 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright 2013 John Wiley & Sons, Ltd. PMID:24027138

  13. 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

  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. PMID:24449458

  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. 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

  17. 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. PMID:25449917

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

    PubMed

    Miyoshi, Hirotoshi; Ohshima, Norio; Sato, Chiaki

    2013-01-01

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

  19. Bone marrow stromal cells and their use in regenerating bone.

    PubMed

    Cancedda, Ranieri; Mastrogiacomo, Maddalena; Bianchi, Giordano; Derubeis, Anna; Muraglia, Anita; Quarto, Rodolfo

    2003-01-01

    Tissue engineering approaches have recently been devised to repair large bone losses. Tissue engineering takes advantages of the combined use of cultured living cells and 3D scaffolds to deliver vital cells to the damaged site of the patient. Cultured bone marrow stromal cells (BMSCs) can be regarded as a mesenchymal progenitor/precursor cell population derived from adult stem cells. When implanted in immunodeficient mice, BMSCs combined with mineralized 3D scaffolds to form a primary bone tissue that is highly vascularized. We have used autologous BMSC/bioceramic composites to treat full-thickness gaps of tibial diaphysis in sheep. The healing process has been investigated. The sequence of events is as follows: (1) bone formation on the outer surface of the implant; (2) bone formation in the inner cylinder canal; (3) formation of fissures and cracks in the implant body; (4) bone formation in the bioceramic pores. Similar composites whose size and shape reflected each bone defect have been implanted at the lesion sites of three patients. External fixation was used. Patients have been followed for more than three years. The results obtained are very promising and we propose the use of culture-expanded osteoprogenitor cells in conjunction with hydroxyapatite bioceramics as a significant improvement in the repair of critical size long bone defects. PMID:12708654

  20. Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy analysis of titanium surfaces cultured with osteoblast-like cells derived from human mandibular bone.

    PubMed

    Mustafa, Kamal; Pan, Jinshan; Wroblewski, Joanna; Leygraf, Christofer; Arvidson, Kristina

    2002-03-15

    Variations in the oxide films on titanium surfaces blasted with TiO(2) particles of various sizes were analyzed after cultures with cells derived from human mandibular bone. Turned titanium surfaces and surfaces blasted with 63-90-, 106-180-, and 180-300-microm TiO(2) particles were cultured with osteoblast-like cells. The surfaces were characterized before and after the cell culture with electrochemical impedance spectroscopy (EIS). The surface chemical composition of selected samples was analyzed with X-ray photoelectron spectroscopy (XPS). EIS revealed that with respect to the turned surfaces, the effective surface area was about 5, 6, and 4 times larger on the surfaces blasted with 63-90-, 106-180-, and 180-300-microm particles, respectively. After 28 days of the cell culture, the corrosion resistance on all sample types was unaffected. The impedance characteristics suggest a considerable effect of ion incorporation and precipitation during culturing. XPS revealed that before the cell culture, a typical surface layer consisted of TiO(2). After the culture, the surface oxide film contained both phosphorus and calcium, along with large amounts of oxidized carbon (carbonate) and nitrogen. There were lower concentrations of carbon and nitrogen on the blasted surfaces. We concluded that the effective surface area was several times higher on blasted surfaces than on turned surfaces. Cells derived from human mandibular bone affected ion incorporation into the implant surface. PMID:11774327

  1. 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.

  2. 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

  3. 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

  4. 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

  5. Bone-Immune Cell Crosstalk: Bone Diseases

    PubMed Central

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

    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

  6. Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures

    PubMed Central

    Zappitelli, Tanya; Chen, Frieda; Aubin, Jane E.

    2015-01-01

    Gja1Jrt/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1Jrt/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1Jrt/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1Jrt/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1Jrt/+ osteoblasts and increased Pparg2 expression in bone marrow–derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1Jrt/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1Jrt/+ mouse model. PMID:25568340

  7. Culture system for bone metabolic studies

    NASA Astrophysics Data System (ADS)

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

    2005-10-01

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

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

    PubMed

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

    2008-08-01

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

  9. 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

  10. Rat bone marrow-derived mast cells co-cultured with 3T3 fibroblasts in the absence of T-cell derived cytokines require stem cell factor for their survival and maintain their mucosal mast cell-like phenotype.

    PubMed Central

    MacDonald, A J; Thornton, E M; Newlands, G F; Galli, S J; Moqbel, R; Miller, H R

    1996-01-01

    When cultured without fibroblasts, rat bone marrow-derived mast cells (BMMC) contain abundant rat mast cell proteinase type II (RMCP-II), and exhibit survival and proliferation when maintained in mesenteric lymph node conditioned medium (CM). When BMMC were co-cultured with 3T3 fibroblasts in the absence of CM, BMMC numbers increased for 7 days and the BMMC survived for up to 23 days. There was a gradual loss of stored RMCP-II in BMMC that were co-cultured with 3T3 cells, but the fibroblast microenvironment did not induce a detectable increase in the low levels of the connective tissue mast cell (CTMC)-associated proteinase, RMCP-I, in the BMMC. Nor did 3T3 cell co-culture induce significant heparin synthesis in BMMC as judged by the cells' reactivity with the fluorescent heparin-binding dye, berberine sulphate. These results suggest that rat BMMC, unlike murine BMMC, do not have the potential to develop multiple CTMC-like characteristics upon co-culture with 3T3 cells. However, when BMMC and fibroblast co-cultures were treated with an antibody to recombinant rat stem cell factor (rrSCF), mast cell survival was completely abrogated. This result suggests that endogenous, fibroblast-derived SCF is essential for the maintenance of rat BMMC viability in the absence of CM. On the other hand, prior treatment of the fibroblasts with the anti-rrSCF antibody did not affect the adherence of BMMC to the monolayer, implying that (an) other molecule(s) is(are) involved in the attachment process. The demonstration that rat BMMC survival on fibroblasts in vitro is dependent upon SCF may indicate an important mechanism by which tissue mucosal cells can be maintained in vivo in the absence of T-cell derived factors. Images Figure 3 PMID:8774353

  11. 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 23 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5105 cell pellets, 5105 cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-?1 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 5105 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

  12. 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. PMID:22304383

  13. 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.

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

    PubMed Central

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

    2014-01-01

    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 (0.5–1.0×108) 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 cm3 to 57.7±10.6 cm3. The average clinical score according to the Japan Orthopaedic Association improved from 65.6±25.5 points to 87.9±19.0 points. One severe adverse event was observed, which was not related to the clinical trial. Although the efficacy of cell transplantation was still to be determined, all procedures were successfully performed and some young patients with extensive necrotic lesions with pain demonstrated good bone regeneration with amelioration of symptoms. Further improvements in our method using MSCs and the proper selection of patients will open a new approach for the treatment of this refractory disease. PMID:24593258

  15. 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 the treatment of this refractory disease. PMID:24593258

  16. 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; Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Melbourne 3800

    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.

  17. 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

  18. Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation.

    PubMed

    Oliveira, J M; Kotobuki, N; Tadokoro, M; Hirose, M; Mano, J F; Reis, R L; Ohgushi, H

    2010-05-01

    Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10(-8) M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quantified, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be beneficial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation. PMID:20152952

  19. In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells, employing a two-chambered co-culture well design.

    PubMed

    Chen, Kelei; Ng, Kian Siang; Ravi, Sujata; Goh, James C H; Toh, Siew Lok

    2016-04-01

    The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23495238

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

    PubMed Central

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

    2010-01-01

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

  1. 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.

  2. Microarray and proteomic analysis of breast cancer cell and osteoblast co-cultures: role of osteoblast matrix metalloproteinase (MMP)-13 in bone metastasis.

    PubMed

    Morrison, Charlotte; Mancini, Stephanie; Cipollone, Jane; Kappelhoff, Reinhild; Roskelley, Calvin; Overall, Christopher

    2011-09-30

    Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix. CLIP-CHIP® microarrays identified changes in the complete protease and inhibitor expression profile of the breast cancer and osteoblast cells that were induced upon co-culture. A large increase in osteoblast-derived MMP-13 mRNA and protein was observed. Affymetrix analysis and validation showed induction of MMP-13 was initiated by soluble factors produced by the breast tumor cells, including oncostatin M and the acute response apolipoprotein SAA3. Significant changes in the osteoblast secretomes upon addition of MMP-13 were identified by degradomics from which six novel MMP-13 substrates with the potential to functionally impact breast cancer metastasis to bone were identified and validated. These included inactivation of the chemokines CCL2 and CCL7, activation of platelet-derived growth factor-C, and cleavage of SAA3, osteoprotegerin, CutA, and antithrombin III. Hence, the influence of breast cancer metastases on the bone microenvironment that is executed via the induction of osteoblast MMP-13 with the potential to enhance metastases growth by generating a microenvironmental amplifying feedback loop is revealed. PMID:21784845

  3. GDNF and NT-3 induce progenitor bone mesenchymal stem cell differentiation into neurons in fetal gut culture medium.

    PubMed

    Zhu, Tianqi; Yu, Donghai; Feng, Jiexiong; Wu, Xiaojuan; Xiang, Lei; Gao, Heyun; Zhang, Xueqin; Wei, Mingfa

    2015-03-01

    With the increasing use of bone marrow mesenchymal stem cells (BMSCs) in cell therapies, factors regulating BMSC differentiation have become the interest of current research. In this study, we investigated the effects of glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) on the course of BMSC differentiation. BMSCs were isolated from rat bone marrow and transfected with GDNF and NT-3 genes. Compared to mock-transfected BMSCs, GDNF and NT-3 induced BMSC differentiation to reveal neuron-like characteristics, i.e., the positive expression of neuronal marker MAP-2 and astrocyte marker GFAP, as detected by immunofluorescence assays. Semi-quantitative polymerase chain reaction (PCR) and western blot analyses showed that the increase of expression of GDNF and NT-3 in BMSCs also simultaneously elevated the mRNA expression of NSE, nestin, and MAP-2. Furthermore, the cell patch-clamp test demonstrated that the overexpression of GDNF and NT-3 in BMSCs enhanced voltage-activated potassium currents, implying that BMSCs possess great potential as a cell-based therapeutic candidate to treat neurological diseases. PMID:25301495

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

    PubMed

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

    2010-09-15

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

  5. CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro-osteogenic and pro-inflammatory cues.

    PubMed

    Pontikoglou, Charalampos; Langonné, Alain; Ba, Mamadou Aliou; Varin, Audrey; Rosset, Philippe; Charbord, Pierre; Sensébé, Luc; Deschaseaux, Frédéric

    2016-04-01

    Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200(pos) cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up-regulated in CD200(pos) cells compared to CD200(neg) fraction. At the functional level, CD200(pos) cells were prone to mineralize the extra-cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200(pos) cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down-regulated. As dexamethasone has anti-inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro-inflammatory cytokines interleukin-1β and tumour necrosis factor-α increased CD200 membrane expression but down-regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro-inflammatory or pro-osteogenic, CD200 expression was down-regulated when nuclear-factor (NF)-κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro-inflammatory cytokines through the same pathway: NF-κB. PMID:26773707

  6. 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 was significantly lower than on day 3 and 0 (P < 0.05). CONCLUSION: An alternative cultivation method was developed to improve the MSCs adhesion on FDB, demonstrating that dynamic co-culture provides a superior environment over static conditions. PMID:22468180

  7. 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

  8. Conversion of Normal Ly-1-Positive B-Lineage Cells into Ly-1-Positive Macrophages in Long-Term Bone Marrow Cultures

    PubMed Central

    Katoh, Shigeki; Tominaga, Akira; Migita, Masahiro; Kudo, Akira

    1990-01-01

    We obtained eight different cell lines in the long-term bone marrow culture system that showed a germ-line configuration of the joining (J) region segments of the Ig heavy-chain (IgH) genes. Their surface markers were CD45R+, Ly-1+, Lyb-2+, cIgM-, sIgM-, Ia-, Thy-1-, Mac-1-, and IL-2R (Tac)+. Use of very young mice and the presence of IL-5 were important for preferential promotion of the survival of B-lineage lymphocytes bearing the Ly-1 markers. When we treated two of them (J8 and J10) with 5-azacytidine for 24 h followed by co-culture with stromal cells and IL-.5, they became Ly-1+, sIgM+ B cells, and Ly-1+, Mac-1+ macrophagelike cells, respectively. After other early lymphoid lines (J1, J8, and J13) were maintained by co-culture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene, although only J13 rearranged the κ-light chain gene. Northern blot analysis revealed that these cell lines expressed Cμ-mRNA, and λ5-mRNA, consistent with normal pre-B cells. Intriguingly, J1, J8, and J13 expressed c-fms mRNA constitutively. When J13 cells were co-cultured with ST2 and GM-CSF in place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspecific-esterase-positive, and showed phagocytosis of latex beads. These results support evidence for a close relationship between the myeloid and Ly-1+ B-cell pathways of differentiation, and indicate that our IL- 5-dependent clones are multipotential intermediates in differentiation from pro-B cells to B cells and macrophages. PMID:2136207

  9. GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.

    PubMed

    Helft, Julie; Böttcher, Jan; Chakravarty, Probir; Zelenay, Santiago; Huotari, Jatta; Schraml, Barbara U; Goubau, Delphine; Reis e Sousa, Caetano

    2015-06-16

    Dendritic cells (DCs) are key players in the immune system. Much of their biology has been elucidated via culture systems in which hematopoietic precursors differentiate into DCs under the aegis of cytokines. A widely used protocol involves the culture of murine bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate BM-derived DCs (BMDCs). BMDCs express CD11c and MHC class II (MHCII) molecules and share with DCs isolated from tissues the ability to present exogenous antigens to T cells and to respond to microbial stimuli by undergoing maturation. We demonstrate that CD11c(+)MHCII(+) BMDCs are in fact a heterogeneous group of cells that comprises conventional DCs and monocyte-derived macrophages. DCs and macrophages in GM-CSF cultures both undergo maturation upon stimulation with lipopolysaccharide but respond differentially to the stimulus and remain separable entities. These results have important implications for the interpretation of a vast array of data obtained with DC culture systems. PMID:26084029

  10. An ex vivo rodent mandible culture model for bone repair.

    PubMed

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

    2010-12-01

    To understand fully cellular mechanisms during bone tissue repair and engineering, there is a need to develop reproducible three-dimensional organotypic culture models, whereby cells in their natural extracellular matrix can be manipulated. Limitations in current model systems do not allow for this integrated approach. This study aimed to develop and validate an ex vivo fractured rat mandible model, to investigate specific molecular and cellular processes involved in bone repair. Slices of mandible from 28-day-old male Wistar rats were cultured in Trowel-type cultures at the liquid-gas interface for up to 21 days. Maintenance of cell and tissue architecture and viability was shown within fractured mandible slices during all culture periods. Autoradiographic studies demonstrated that resident cells were actively synthesizing and secreting proteins, and cells of the osteoblast lineage were shown to survive throughout the culture periods. The model was responsive to exogenously added transforming growth factor-β1, with observed increases in cellular migration/proliferation and expression of bone matrix proteins. The ex vivo mandible model developed within this study may represent an ideal system for investigating specific processes of bone repair, as well as a promising alternative to in vivo testing of novel clinical therapeutics. PMID:20218818

  11. Cells isolated from bone-marrow and lungs of allergic BALB/C mice and cultured in the presence of IL-5 are respectively resistant and susceptible to apoptosis induced by dexamethasone.

    PubMed

    Maximiano, Elisabeth S; Elsas, P Xavier; de Mendonça Sales, Simone C; Jones, Carla P; Joseph, Danielle; Vargaftig, B Boris; Gaspar Elsas, Maria Ignez C

    2005-05-01

    We have previously reported that, in IL-5-stimulated bone-marrow cultures, dexamethasone upregulates eosinophil differentiation and protects developing eosinophils from apoptosis induced by a variety of agents. Recently developed procedures for the isolation of hemopoietic cells from allergic murine lungs have enabled us to evaluate how these cells respond to dexamethasone in IL-5-stimulated cultures, when compared with bone-marrow-derived cells isolated from the same donors, and whether differences in response patterns were linked to apoptosis. Ovalbumin challenge of sensitized mice increased significantly the numbers of mature leukocytes as well as hemopoietic cells recovered from digested lung fragments, relative to saline-challenged, sensitized controls. Both mature eosinophils and cells capable of differentiating into eosinophils in the presence of IL-5 were present in lungs from sensitized mice 24 h after airway challenge. Dexamethasone strongly inhibited eosinophil differentiation in IL-5-stimulated cultures of lung hemopoietic cells. By contrast, dexamethasone enhanced eosinophil differentiation in cultures of allergic bone-marrow cells, in identical conditions. Hemopoietic cells from lungs and bone-marrow were respectively susceptible and resistant to induction of apoptosis by dexamethasone. The dexamethasone-sensitive step was the response to IL-5 in culture, while accumulation of IL-5 responsive cells in allergen-challenged lungs was dexamethasone-resistant. Cells from lungs and bone-marrow, cultured for 3 days with IL-5 in the absence of dexamethasone, did not respond to a subsequent exposure to dexamethasone in the presence of IL-5. These findings confirm that IL-5-responsive hemopoietic cells found in challenged, sensitized murine lungs differ from those in bone-marrow, with respect to the cellular responses induced by dexamethasone, including apoptosis. PMID:15778122

  12. Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

    PubMed Central

    Yoshida, N; Radbruch, A; Rajewsky, K

    1987-01-01

    In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2824191

  13. 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

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

    SciTech Connect

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

    1982-12-01

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

  15. Bone scaffold architecture modulates the development of mineralized bone matrix by human embryonic stem cells

    PubMed Central

    Marcos-Campos, Ivan; Marolt, Darja; Petridis, Petros; Bhumiratana, Sarindr; Schmidt, Daniel; Vunjak-Novakovic, Gordana

    2012-01-01

    Decellularized bone has been widely used as a scaffold for bone formation, due to its similarity to the native bone matrix and excellent osteoinductive and biomechanical properties. We have previously shown that human mesenchymal and embryonic stem cells form functional bone matrix on such scaffolds, without the use of growth factors. In this study, we focused on differences in bone matrix that exist even among identical harvesting sites, and the effects of the matrix architecture and mineral content on bone formation by human embryonic stem cells (hESC). Mesenchymal progenitors derived from hESCs were cultured for 5 weeks in decellularized bone scaffolds with three different densities: low (0.281 ± 0.018 mg/mm3), medium (0.434 ± 0.015 mg/mm3) and high (0.618 ± 0.027 mg/mm3). The medium-density group yielded highest densities of cells and newly assembled bone matrix, presumably due to the best balance between the transport of nutrients and metabolites to and from the cells, space for cell infiltration, surface for cell attachment and the mechanical strength of the scaffolds, all of which depend on the scaffold density. Bone mineral was beneficial for the higher expression of bone markers in cultured cells and more robust accumulation of the new bone matrix. PMID:22901965

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

    PubMed

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

    2005-03-01

    Although the function and effects of many growth factors and extracellular matrix (ECM) molecules have been described for several periodontal tissues in vivo and in vitro, the molecular interactions involved in the communication between cells of the periodontal ligament and the alveolar bone are poorly understood. To contribute to the identification of such interactions, we have generated co-cultures (CCs) of periodontal ligament fibroblasts (PDLs) and alveolar bone cells (ABCs) and compared mRNA expression for various growth factors, ECM molecules, and matrix metalloproteinase13 (MMP13) after 1 and 2 weeks with matched mono-cultures (MCs) by reverse transcription/polymerase chain reaction. Compared with CCs of 1 week, PDLs and ABCs after 2 weeks revealed relatively high levels of all analyzed mRNAs, viz., for EGF, HGF, VEGF, TGFbeta1, collagen-I (COL1), osteonectin (ON), fibronectin (FN1), and MMP13. At week 2, when compared with MCs, CCs showed an elevation of all tested mRNAs in PDLs and ABCs, except for TGFbeta1 and FN1, which only increased in PDLs. After 1 week, when CCs were compared with MCs, mRNAs for HGF and TGFbeta1 were less abundant in PDLs and ABCs, whereas the other genes exhibited lower expression levels in only one of the cell types. Analysis of our data indicated that the expression of mRNAs for growth factors and for COL1, ON, FN1, and MMP13 was modulated in the distinct cell types with respect to culture time and culture type. The differences in the mRNA expression patterns between CCs and MCs suggest that the respective genes are involved in the molecular interactions of PDLs and ABCs. PMID:15668800

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

    PubMed Central

    2014-01-01

    Background Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. Methods We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients’ primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. Results Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. Conclusions Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies. PMID:24947048

  18. Freshly Thawed and Continuously Cultured Human Bone Marrow-Derived Mesenchymal Stromal Cells Comparably Ameliorate Allergic Airways Inflammation in Immunocompetent Mice

    PubMed Central

    Cruz, Fernanda F.; Borg, Zachary D.; Goodwin, Meagan; Sokocevic, Dino; Wagner, Darcy; McKenna, David H.; Rocco, Patricia R.M.

    2015-01-01

    Recent data suggest that freshly thawed previously frozen mesenchymal stromal cells (MSCs) may not have the same effectiveness or breadth of anti-inflammatory activities as do continuously cultured MSCs. This has significant implications for clinical use, in which many infusion schemes use frozen cells thawed at the bedside for administration. The available data, however, predominantly evaluate in vitro MSC properties, and so far there has been limited in vivo analysis. To further assess this issue, we compared freshly thawed (thawed) versus continuously cultured (fresh) human bone marrow-derived MSC (hMSC) administration in a mouse model of mixed Th2/Th17 allergic airway inflammation induced by Aspergillus hyphal extract (AHE) exposures in immunocompetent C57Bl/6 mice. Control cell populations included fresh versus thawed murine bone marrow-derived MSCs (mMSCs) and human lung fibroblasts (HLFs). Systemic administration of both thawed and fresh hMSCs and mMSCs, but not HLFs, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE-provoked increases in airway hyper-reactivity, lung inflammation, and antigen-specific CD4 T-cell Th2 and Th17 phenotype. Notably, there was no difference in effects of fresh versus thawed hMSCs or mMSCs on any outcome measured except for some variability in the effects on the bronchoalveolar lavage fluid composition. These results demonstrated potent xenogeneic effects of human MSCs in an immunocompetent mouse model of allergic airways inflammation and that thawed MSCs are as effective as fresh MSCs. The question of fresh versus thawed MSC effectiveness needs to be investigated carefully and may differ in different in vivo disease-specific models. Significance This study addressed whether freshly thawed mesenchymal stromal cells (MSCs) are as effective in in vivo settings as those that have been continuously cultured. It also provided further data demonstrating that xenogeneic use of MSCs in immunocompetent mice is as effective as murine MSCs. This information provides further support and direction for potential clinical use of MSCs in patients with severe asthma. PMID:25925837

  19. 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 on hPDLCs than the inorganic elements. PMID:25738431

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

    PubMed Central

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

    2010-01-01

    Background Most forms of chronic kidney disease are characterized by progressive renal and cardiac fibrosis leading to dysfunction. Preliminary evidence suggests that various bone marrow-derived cell populations have antifibrotic effects. In exploring the therapeutic potential of bone marrow derived cells in chronic cardio-renal disease, we examined the anti-fibrotic effects of bone marrow-derived culture modified cells (CMCs) and stromal cells (SCs). Methodology/Principal Findings In vitro, CMC-conditioned medium, but not SC-conditioned medium, inhibited fibroblast collagen production and cell signalling in response to transforming growth factor-ß. The antifibrotic effects of CMCs and SCs were then evaluated in the 5/6 nephrectomy model of chronic cardio-renal disease. While intravascular infusion of 106 SCs had no effect, 106 CMCs reduced renal fibrosis compared to saline in the glomeruli (glomerulosclerosis index: 0.8±0.1 v 1.9±0.2 arbitrary units) and the tubulointersitium (% area type IV collagen: 1.2±0.3 v 8.4±2.0, p<0.05 for both). Similarly, 106 CMCs reduced cardiac fibrosis compared to saline (% area stained with picrosirius red: 3.2±0.3 v 5.1±0.4, p<0.05), whereas 106 SCs had no effect. Structural changes induced by CMC therapy were accompanied by improved function, as reflected by reductions in plasma creatinine (58±3 v 81±11 µmol/L), urinary protein excretion (9×/÷1 v 64×/÷1 mg/day), and diastolic cardiac stiffness (left ventricular end-diastolic pressure-volume relationship: 0.030±0.003 v 0.058±0.011 mm Hg/µL, p<0.05 for all). Despite substantial improvements in structure and function, only rare CMCs were present in the kidney and heart, whereas abundant CMCs were detected in the liver and spleen. Conclusions/Significance Together, these findings provide the first evidence suggesting that CMCs, but not SCs, exert a protective action in cardio-renal disease and that these effects may be mediated by the secretion of diffusible anti-fibrotic factor(s). PMID:20209052

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

    PubMed

    Lehmann, Tomasz P; Filipiak, Krystyna; Juzwa, Wojciech; Sujka-Kordowska, Patrycja; Jagodziński, Paweł P; Zabel, Maciej; Głowacki, Jakub; Misterska, Ewa; Walczak, Michał; Głowacki, Maciej

    2014-02-01

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

  2. [Use of cultured human autologous bone marrow stem cells in repair of a rotator cuff tear: preliminary results of a safety study].

    PubMed

    Havlas, V; Kotaška, J; Koníček, P; Trč, T; Konrádová, Š; Kočí, Z; Syková, E

    2015-01-01

    PURPOSE OF THE STUDY Rotator cuff tears are one of the most frequent shoulder disorders which are often associated with pain and interfere with proper arm function. In order to evaluate the safety and effectiveness of using cultured human autologous mesenchymal stem cells (MSC) applied to the suture site during arthroscopic repair of a rotator cuff tear, a prospective clinical study was designed and started recently at the authors' department. Its primary goal was to evaluate the safety of using cultured human MSCs, the secondary goal then was to study a therapeutic effect of their application. Preliminary results of the study on a limited number of patients are presented here. MATERIAL AND METHODS Ten patients who met the indication criteria for arthroscopic repair of a rotator cuff tear were included in the study. In addition, they also had to meet inclusion and lack exclusion criteria. According to the protocol, their bone marrow was harvested at 3 to 4 weeks before surgery. Subsequently, an arthroscopic repair of the rotator cuff tear was performed and an suspension of cultured MSCs was applied to the suture site at the end of the procedure. The isolation of MSCs from bone marrow and their cultivation was carried out by the company Bioinova, Ltd. The patients were followed up at 6 weeks and 3 and 6 months post-operatively. Their clinical assessment included physical examination of the shoulder, pain intensity evaluation according to the visual analogue scale (VAS), and subjective questionnaires for Constant and University of California (UCLA) scores. All patients underwent MRI examination at 6 post-operative months to evaluate the quality of rotator cuff reconstruction. The findings were compared with the pre-operative results. RESULTS A final evaluation was made in eight patients of 10. Two patients were excluded from the study because their exclusion criteria were fulfilled. The evaluated patients showed significantly better clinical outcomes as early as 6 weeks after surgery; also all pre-operative scores were improved at 3 and 6 months. The average values at 6 months post-operatively were: 0 points for the VAS score, 32 for the UCLA score and 84 for the Constant score. The MRI findings at 6 months after surgery showed fully healed and well-integrated tissue of the rotator cuff tendon attachment in all eight patients. No adverse effects of therapy were recorded during the follow-up period. DISCUSSION The use of autologous stem cells and growth factors in the treatment of tendons, muscles and cartilage is currently the topic of many experimental studies on animal models. Its utilisation in human clinical trials has been reported only marginally; the relevant studies have so far used only suspensions of non-cultured mononuclear cells. Our study, although on a smallsize patient group, provides evidence that human cultured autologous MSCs can safely be used for tissue repair in the indications mentioned above. CONCLUSIONS Our preliminary short-term results show that using human cultured autologous MSCs in the treatment of rotator cuff tears is safe. However, further research is needed, particularly with regard to the effectiveness of the method. Key words: rotator cuff tear, arthroscopic repair, mesenchymal stem cells, tendon, cell therapy. PMID:26317295

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

    PubMed Central

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

    2009-01-01

    The tremendous need for bone tissue in numerous clinical situations and the limited availability of suitable bone grafts are driving the development of tissue engineering approaches to bone repair. In order to engineer viable bone grafts, one needs to understand the mechanisms of native bone development and fracture healing, as these processes should ideally guide the selection of optimal conditions for tissue culture and implantation. Engineered bone grafts have been shown to have capacity for osteogenesis, osteoconduction, osteoinduction and osteointegration - functional connection between the host bone and the graft. Cells from various anatomical sources in conjunction with scaffolds and osteogenic factors have been shown to form bone tissue in vitro. The use of bioreactor systems to culture cells on scaffolds before implantation further improved the quality of the resulting bone grafts. Animal studies confirmed the capability of engineered grafts to form bone and integrate with the host tissues. However, the vascularization of bone remains one of the hurdles that need to be overcome if clinically sized, fully viable bone grafts are to be engineered and implanted. We discuss here the biological guidelines for tissue engineering of bone, the bioreactor cultivation of human mesenchymal stem cells on three-dimensional scaffolds, and the need for vascularization and functional integration of bone grafts following implantation. PMID:19075755

  4. siDNMT1 Increases γ-globin Expression in Chemical-Inducer-of-Dimerization (CID)-Dependent Mouse βYAC Bone Marrow Cells and in Baboon Erythroid Progenitor Cell Cultures

    PubMed Central

    Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; Ruiz, Maria Armila; Peterson, Kenneth; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult stage erythroid cells. 2) Methods DNMT1 levels were reduced by nucleofection of siRNA targeting DNMT1 in chemical-inducer-of-dimerization (CID)-dependent multipotential mouse bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and in primary cultures of erythroid progenitor cells derived from CD34+ baboon BM cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real time PCR and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabelling of globin chains followed by HPLC analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. 3) Results Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin mRNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. 4) Conclusion DNMT1 is required to maintain DNA methylation of the γ-globin gene promoters and repress γ-globin gene expression in adult-stage erythroid cells. PMID:20974210

  5. 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

  6. Chromatin Changes at the PPAR-γ2 Promoter During Bone Marrow-Derived Multipotent Stromal Cell Culture Correlate With Loss of Gene Activation Potential.

    PubMed

    Lynch, Patrick J; Thompson, Elaine E; McGinnis, Kathleen; Rovira Gonzalez, Yazmin I; Lo Surdo, Jessica; Bauer, Steven R; Hursh, Deborah A

    2015-07-01

    Bone marrow-derived multipotent stromal cells (BM-MSCs) display a broad range of therapeutically valuable properties, including the capacity to form skeletal tissues and dampen immune system responses. However, to use BM-MSCs in a clinical setting, amplification is required, which may introduce epigenetic changes that affect biological properties. Here we used chromatin immunoprecipitation to compare post-translationally modified histones at a subset of gene promoters associated with developmental and environmental plasticity in BM-MSCs from multiple donors following culture expansion. At many locations, we observed localization of both transcriptionally permissive (H3K4me3) and repressive (H3K27me3) histone modifications. These chromatin signatures were consistent among BM-MSCs from multiple donors. Since promoter activity depends on the relative levels of H3K4me3 and H3K27me3, we examined the ratio of H3K4me3 to H3K27me3 (K4/K27) at promoters during culture expansion. The H3K4me3 to H3K27me3 ratios were maintained at most assayed promoters over time. The exception was the adipose-tissue specific promoter for the PPAR-γ2 isoform of PPAR-γ, which is a critical positive regulator of adipogenesis. At PPAR-γ2, we observed a change in K4/K27 levels favoring the repressed chromatin state during culture. This change correlated with diminished promoter activity in late passage cells exposed to adipogenic stimuli. In contrast to BM-MSCs and osteoblasts, lineage-restricted preadipocytes exhibited levels of H3K4me3 and H3K27me3 that favored the permissive chromatin state at PPAR-γ2. These results demonstrate that locus-specific changes in H3K4me3 and H3K27me3 levels can occur during BM-MSC culture that may affect their properties. Stem Cells 2015;33:2169-2181. PMID:25640287

  7. 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.

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

    PubMed

    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. PMID:3292279

  9. Resorbing bone stimulates tumor cell growth. A role for the host microenvironment in bone metastasis.

    PubMed

    Manishen, W J; Sivananthan, K; Orr, F W

    1986-04-01

    Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for Walker tumor cells, the TE-85 osteosarcoma, and human breast carcinoma lines. These results suggest a mechanism by which bone resorption could promote the development of skeletal metastasis. PMID:3457536

  10. 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 cells compared with that of gene transfection alone. Furthermore, significantly enhanced reparative effects were observed in the myocardium of rabbits following treatment with Nkx2.5- or GATA-4-transfected myocardial cell extracellular environment co-cultured BMSCs compared with those treated with untreated BMSCs. PMID:25975979

  11. The Expression of Bone Morphogenetic Protein 2 and Matrix Metalloproteinase 2 through Retinoic Acid Receptor Beta Induced by All-Trans Retinoic Acid in Cultured ARPE-19 Cells

    PubMed Central

    Gao, Zhenya; Huo, Lijun; Cui, Dongmei; Yang, Xiao; Zeng, Junwen

    2016-01-01

    Purpose All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells. Methods The effects of ATRA (concentrations from 10−9 to 10−5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10−9 to 10−5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ. Results RARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10−9 to 10−5 mol/l) with a maximum effect observed at 10−6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10−6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135. Conclusion ATRA induced upregulation of RARβ in ARPE-19 cells and stimulated these cells to secrete BMP-2 and MMP-2. PMID:26967733

  12. 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

  13. 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…

  14. Optimizing stem cell culture

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2013-01-01

    Adrenocorticotropic hormone (ACTH) is among the several melanocortin peptide hormones that are derived from proopiomelanocortin (POMC). ACTH has been found to enhance osteogenesis and chondrogenesis. We show that in the presence of dexamethasone, ACTH dose-dependently increases chondrogenic nodule formation in bone marrow stromal cells (BMSC) from the Wystar Kyoto (WKY) rat. The nodules consist of condensed cells highly expressing alkaline phosphatase, Sox9 and type II collagen transcripts, and a proteoglycan rich matrix. Immunoblot analysis of crude membrane fractions was used to determine that these cells express three melanocortin receptors (MC-R); MC2-R, MC3-R and MC5-R, as well as the melanocortin 2-receptor accessory protein (MRAP). To determine which of these receptors mediate ACTH-induced effects, we used MC-R specific peptides and the known agonist profiles of the receptors. Neither ?-MSH, a strong agonist of the MC5-R nor ?2-MSH, a strong agonist of the MC3-R, duplicates ACTH effects in rat BMSC. In addition, calcium flux was examined as a mechanism for ACTH action at the MC2-R. Consistent with MC2-R and MRAP expression patterns in the BMSC cultures, ACTH-induced transient increases in intracellular calcium were increased with dexamethasone treatment. Neither ?-MSH nor ?2-MSH affected calcium flux. Dexamethasone increased MC2-R and MRAP expression as well as POMC peptide expression and cleavage to increase the production of the lipolytic ?-LPH product. Therefore the effects of ACTH in rat BMSC enriched for mesenchymal progenitors are consistent with an MC2-R signaling mechanism and dexamethasone is capable of regulating components of the melanocortin system in these cells. PMID:23358747

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

    PubMed

    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. PMID:27054380

  17. 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.

  18. Indirect co‑culture of vascular smooth muscle cells with bone marrow mesenchymal stem cells inhibits vascular calcification and downregulates the Wnt signaling pathways.

    PubMed

    Zhu, Meng'en; Fang, Xin; Zhou, Shaoqiong; Li, Wei; Guan, Siming

    2016-06-01

    Vascular calcification (VC) is widely considered to be a crucial clinical indicator of cardiovascular disease. Recently, certain properties of mesenchymal stem cells (MSCs) have been hypothesized to have potential in treating cardiovascular diseases. However, their effect on the initiation and progression of VC remains controversial. The present study aimed to investigate whether MSCs indirectly mediate VC and their impact on the Wnt signaling pathways. A Transwell system was selected to establish the indirect co‑culture environment, and hence, vascular smooth muscle cells (VSMCs) were indirectly co‑cultured in the presence or absence of MSCs at a ratio of 1:1. Osteogenic medium (OS) was added to imitate a calcifying environment. Fourteen days later, VSMCs in the lower layers of the Transwell plates were harvested. Alkaline phosphatase activity and calcium nodules were markedly increased in calcific VSMCs induced by OS. However, these parameters were significantly decreased in VSMCs by indirectly co‑culturing with MSCs in the same medium. Furthermore, the messenger RNA expression levels of osteopontin and osteoprotegerin were notably increased in VSMCs cultured in OS, but reduced by indirect interaction with MSCs. In addition, the activities of canonical and noncanonical Wnt ligands, wingless‑type MMTV integration site family, number 5A (Wnt5a), receptor tyrosine kinase‑like orphan receptor 2 (Ror2) and β‑catenin, which are important in the process of VC, were downregulated by indirect contact with MSCs in OS. Thus, indirect co‑culture with MSCs inhibits VC and downregulates the Wnt signaling pathways. PMID:27121342

  19. Effect of naringin on bone cells.

    PubMed

    Wong, R W K; Rabie, A B M

    2006-11-01

    Statin, a HMG-CoA reductase inhibitor, was shown to increase BMP-2 gene expression for bone formation, by blocking the mevalonate pathway in cholesterol production. We investigated the effect of naringin, a flavonoid available commonly in citrus fruits, which was also a HMG-CoA reductase inhibitor, in UMR 106 osteoblastic cell line in vitro. The control group consisted of cells cultured without any intervention for different time intervals (24 h, 48 h, and 72 h), whereas the experimental (naringin) group consisted of cells cultured with naringin of different concentrations (0.001 micromol/L, 0.01 micromol/L, and 0.1 micromol/L) for the same time intervals of the control. Colorimetric Tetrazolium (MTT) assay, total protein content assay, and alkaline phosphatase activity were used to measure the cellular activities. Results for the naringin group showed an increase in MTT assay compared with the control and the effect was dose dependent. At high concentration (0.1 micromol), the increases ranged from 60% to 80%. In the total protein content assay, naringin also showed an increase compared with control and the effect was also dose dependent. At high concentration (0.1 micromol), the increases ranged from 9% to 20%. In the alkaline phosphatase activity assay, naringin at high concentration (0.1 micromol) significantly increased the activity up to 20%. In conclusion, naringin significantly increased bone cell activities in vitro. This is the first study specifically attempted to investigate the effect of naringin on bone cell activities. Besides statin, this provided another example of mevalonate pathway blockage in the cholesterol production pathway by HMG-CoA reductase inhibition will increase the bone cell activities. PMID:16944474

  20. Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering.

    PubMed

    Ghoniem, Amir-Alexander; Açil, Yahya; Wiltfang, Jörg; Gierloff, Matthias

    2015-06-01

    To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation. PMID:26140219

  1. 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.; Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, FIN-90014 Oulu ; 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.

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

    PubMed

    Miettinen, Johanna A; Pietilä, 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-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α 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-α 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-α 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-α exposure, which might influence MSC differentiation stage and capacity. PMID:21182837

  3. Cell isolation and culture.

    PubMed Central

    Zhang, Sihui; Kuhn, Jeffrey R

    2013-01-01

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

  4. [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. PMID:24870836

  5. Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth.

    PubMed

    Jing, Y; Zhou, X; Han, X; Jing, J; von der Mark, K; Wang, J; de Crombrugghe, B; Hinton, R J; Feng, J Q

    2015-12-01

    For decades, it has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation. However, very recent studies in long bone suggest that chondrocytes can directly transform into bone cells. Our initial in vivo characterization of condylar hypertrophic chondrocytes revealed modest numbers of apoptotic cells but high levels of antiapoptotic Bcl-2 expression, some dividing cells, and clear alkaline phosphatase activity (early bone marker). Ex vivo culture of newborn condylar cartilage on a chick chorioallantoic membrane showed that after 5 d the cells on the periphery of the explants had begun to express Col1 (bone marker). The cartilage-specific cell lineage-tracing approach in triple mice containing Rosa 26(tdTomato) (tracing marker), 2.3 Col1(GFP) (bone cell marker), and aggrecan Cre(ERT2) (onetime tamoxifen induced) or Col10-Cre (activated from E14.5 throughout adult stage) demonstrated the direct transformation of chondrocytes into bone cells in vivo. This transformation was initiated at the inferior portion of the condylar cartilage, in contrast to the initial ossification site in long bone, which is in the center. Quantitative data from the Col10-Cre compound mice showed that hypertrophic chondrocytes contributed to ~80% of bone cells in subchondral bone, ~70% in a somewhat more inferior region, and ~40% in the most inferior part of the condylar neck (n = 4, P < 0.01 for differences among regions). This multipronged approach clearly demonstrates that a majority of chondrocytes in the fibrocartilaginous condylar cartilage, similar to hyaline cartilage in long bones, directly transform into bone cells during endochondral bone formation. Moreover, ossification is initiated from the inferior portion of mandibular condylar cartilage with expansion in one direction. PMID:26341973

  6. Mesenchymal Stem Cells for Bone Repair and Metabolic Bone Diseases

    PubMed Central

    Undale, Anita H.; Westendorf, Jennifer J.; Yaszemski, Michael J.; Khosla, Sundeep

    2009-01-01

    Human mesenchymal stem cells offer a potential alternative to embryonic stem cells in clinical applications. The ability of these cells to self-renew and differentiate into multiple tissues, including bone, cartilage, fat, and other tissues of mesenchymal origin, makes them an attractive candidate for clinical applications. Patients who experience fracture nonunion and metabolic bone diseases, such as osteogenesis imperfecta and hypophosphatasia, have benefited from human mesenchymal stem cell therapy. Because of their ability to modulate immune responses, allogeneic transplant of these cells may be feasible without a substantial risk of immune rejection. The field of regenerative medicine is still facing considerable challenges; however, with the progress achieved thus far, the promise of stem cell therapy as a viable option for fracture nonunion and metabolic bone diseases is closer to reality. In this review, we update the biology and clinical applicability of human mesenchymal stem cells for bone repair and metabolic bone diseases. PMID:19797778

  7. Bone regeneration: the stem/progenitor cells point of view.

    PubMed

    Deschaseaux, Frédéric; Pontikoglou, Charalampos; Sensébé, Luc

    2010-01-01

    After bone injuries, several molecular mechanisms establish bone repair from stem/progenitor cells. Inflammation factors attract regenerative cells which expand and differentiate in order to build up a bone highly similar to that before injury. Bone marrow (BM) mesenchymal stem cells (MSCs) as skeletal stem cells and endothelial progenitors (EPCs) are at the origin of such reparation mechanisms. However, discrepancies exist about their identities. Although cultured MSCs are extensively described, their in vivo native forms are poorly known. In addition, recent experiments show that several types of EPC exist. We therefore review up-to-date data on the characterization of such stem/progenitor cells and propose a new point of view of their function in bone regeneration. PMID:19840188

  8. Effects of ionizing radiation on differentiation of murine bone marrow cells into mast cells

    PubMed Central

    Murakami, Sho; Yoshino, Hironori; Ishikawa, Junya; Yamaguchi, Masaru; Tsujiguchi, Takakiyo; Nishiyama, Ayaka; Yokoyama, Kouki; Kashiwakura, Ikuo

    2015-01-01

    Mast cells, immune effector cells produced from bone marrow cells, play a major role in immunoglobulin E–mediated allergic responses. Ionizing radiation affects the functions of mast cells, which are involved in radiation-induced tissue damage. However, whether ionizing radiation affects the differential induction of mast cells is unknown. Here we investigated whether bone marrow cells of X-irradiated mice differentiated into mast cells. To induce mast cells, bone marrow cells from X-irradiated and unirradiated mice were cultured in the presence of cytokines required for mast cell induction. Although irradiation at 0.5 Gy and 2 Gy decreased the number of bone marrow cells 1 day post-irradiation, the cultured bone marrow cells of X-irradiated and unirradiated mice both expressed mast cell–related cell-surface antigens. However, the percentage of mast cells in the irradiated group was lower than in the unirradiated group. Similar decreases in the percentage of mast cells induced in the presence of X-irradiation were observed 10 days post irradiation, although the number of bone marrow cells in irradiated mice had recovered by this time. Analysis of mast cell function showed that degranulation of mast cells after immunoglobulin E–mediated allergen recognition was significantly higher in the X-irradiated group compared with in the unirradiated group. In conclusion, bone marrow cells of X-irradiated mice differentiated into mast cells, but ionizing radiation affected the differentiation efficiency and function of mast cells. PMID:26453633

  9. 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

  10. Adipose-derived stem cells combined with a demineralized cancellous bone substrate for bone regeneration.

    PubMed

    Shi, Yaling; Niedzinski, Jerry R; Samaniego, Adrian; Bogdansky, Simon; Atkinson, Brent L

    2012-07-01

    Mesenchymal stem cells (MSCs) isolated from cadaveric adipose tissue can be obtained in large quantities, and have been reported in the literature to be capable of inducing bone formation in vivo and ex vivo.( 1-6 ) The hypothesis tested whether a demineralized cancellous bone matrix (DCBM) can provide an effective substrate for selection and retention of stem cells derived from the stromal vascular fraction (SVF) of adipose. Human cadaveric adipose tissue was recovered from a donor and digested. The resulting SVF-containing MSCs were seeded onto the demineralized bone allografts, after which the nonadherent cells were washed off. The MSCs were characterized using a flow cytometer and tri-lineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) in vitro. The stem cell-seeded allografts were also characterized for cell number, adherence to the DCBM, osteogenic activity (alkaline phosphatase and Alizarin Red staining), and bone morphorgenic protein (BMP) quantity. Flow cytometry identified a mean total of 7.2% MSCs in SVF and 87.2% MSCs after culture. The stem cells showed the capability of differentiating into bone, cartilage, and fat. On the 21 stem cell-seeded bone allografts, there were consistent, attached, viable cells (100,74422,762 cells/cube). An assessment of donor age, gender, and body mass index revealed no significant differences in cell numbers. Enzyme-linked immunosorbent assay revealed the presence of BMP-2 and BMP-7. In conclusion, this bone graft contains three key elements for bone regeneration: adhered osteogenic stem cells, 3D osteoconductive bone scaffold, and osteoinductive BMP signal. It therefore has the potential to be effective for bone regeneration. PMID:22500696

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

    PubMed Central

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

    2011-01-01

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

  12. Confocal fluorescence microscopy: some applications in bone cell biology.

    PubMed

    Jones, S J; Taylor, M L

    1990-05-01

    Three-dimensional information is necessary for the proper investigation of the interrelationships of bone cells, and of the complex interface between these cells and the bone matrix they form and destroy. The use of fluorescence confocal microscopy was explored in the determination of the distribution of immunolabelled actin and vinculin--cytoskeletal and attachment proteins--in isolated chick bone cells cultured on dentine, and in neonate rat and rabbit calvaria. Confocal imaging, compared with conventional fluorescence imaging, greatly enhanced the interpretation possible. PMID:2115085

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

    PubMed

    Zubov, D O

    2008-01-01

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

  14. 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)

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

    PubMed Central

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

    2014-01-01

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

  16. [Thermophilic eukaryotic cell cultures].

    PubMed

    Bakhutashvili, V I; Dzhavakhishvili, N A; Kupradze, S A; Chkhotua, R N; Bobokhidze, N G

    1991-01-01

    Thermophilic clones of lymphoblastoid cell cultures Namalwa were generated and found to be capable of life and reproduction at a temperature of 60 degrees C. The reproductive dynamics, cytology, and ultrastructure of these clones were studied. PMID:1803780

  17. 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. PMID:25297116

  18. 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

  19. Cell interactions in bone tissue engineering

    PubMed Central

    Pirraco, R P; Marques, A P; Reis, R L

    2010-01-01

    Abstract Bone fractures, where the innate regenerative bone response is compromised, represent between 4 and 8 hundred thousands of the total fracture cases, just in the United States. Bone tissue engineering (TE) brought the notion that, in cases such as those, it was preferable to boost the healing process of bone tissue instead of just adding artificial parts that could never properly replace the native tissue. However, despite the hype, bone TE so far could not live up to its promises and new bottom-up approaches are needed. The study of the cellular interactions between the cells relevant for bone biology can be of essential importance to that. In living bone, cells are in a context where communication with adjacent cells is almost permanent. Many fundamental works have been addressing these communications nonetheless, in a bone TE approach, the 3D perspective, being part of the microenvironment of a bone cell, is as crucial. Works combining the study of cell-to-cell interactions in a 3D environment are not as many as expected. Therefore, the bone TE field should not only gain knowledge from the field of fundamental Biology but also contribute for further understanding the biology of bone. In this review, a summary of the main works in the field of bone TE, aiming at studying cellular interactions in a 3D environment, and how they contributed towards the development of a functional engineered bone tissue, is presented. PMID:20050963

  20. Stem Cells and Calcium Phosphate Cement Scaffolds for Bone Regeneration.

    PubMed

    Wang, P; Zhao, L; Chen, W; Liu, X; Weir, M D; Xu, H H K

    2014-07-01

    Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications. PMID:24799422

  1. 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. PMID:23943083

  2. Mechanotransduction in bone: do bone cells act as sensors of fluid flow?

    PubMed

    Turner, C H; Forwood, M R; Otter, M W

    1994-08-01

    When compact bone is subjected to bending loads, interstitial fluid in the bone matrix flows away from regions of high compressive stress. The amount of interstitial fluid flow is strongly influenced by the loading rate in a dose-dependent fashion. We hypothesize that interstitial fluid flow affects bone formation, and we tested this hypothesis indirectly by measuring the effect of different loading frequencies on bone formation rate in vivo. The right tibiae of adult female rats were subjected to applied bending at frequencies of 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 Hz for a 2-wk period. The rats were then killed and histomorphometric measurements of bone formation were made of the midshaft of the tibia. Bending of the tibia increased bone formation rate in the higher-frequency (0.5 to 2.0 Hz) loading groups as much as fourfold, yet no increase in bone formation rate was observed for loading frequencies below 0.5 Hz. In a separate experiment, we found stress-generated potentials (SGP) in the rat tibia to increase monotonically with increasing loading frequency. The dose-response relationship between loading frequency and the bone formation response closely resembles the relationship between loading frequency and SGP within bone. The qualitative similarity between these two relationships suggests that increased bone formation is associated with increased SGP, which are caused by interstitial fluid flow. Bone cells are known to be sensitive to electric fields and may respond directly to SGP. Also, fluid shear forces have been shown to stimulate bone cells in culture, so it is possible that increased interstitial fluid flow directly affects bone formation. PMID:8070637

  3. 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

  4. 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. PMID:23623428

  5. 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. PMID:22532569

  6. 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

  7. Generation of Eosinophils from Cryopreserved Murine Bone Marrow Cells

    PubMed Central

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

    2014-01-01

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

  8. Culturing Uveal Melanoma Cells

    PubMed Central

    Angi, Martina; Versluis, Mieke; Kalirai, Helen

    2015-01-01

    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

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

    PubMed

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

    2000-12-01

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

  10. 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 dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

  11. 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

  12. 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

  13. Inhibiting and stimulating effects of TGF-. beta. 1 on osteoclastic bone resorption in fetal mouse bone organ cultures

    SciTech Connect

    Dieudonne, S.C.; Foo, P.; van Zoelen, E.J.; Burger, E.H. )

    1991-05-01

    The effects of TGF-{beta} 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.

  14. 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. PMID:24637629

  15. 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

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX-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 permanent cell cycle blocks lead to a depletion of proliferation-competent cells from the osteoblastic precursor pool in the body, a gradual decrease of bone mass in weightlessness may be attributed to synergistic effects of radiation and weightlessness.

  16. 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. PMID:23166000

  17. Exogenous regucalcin suppresses osteoblastogenesis and stimulates adipogenesis in mouse bone marrow culture.

    PubMed

    Yamaguchi, Masayoshi; Weitzmann, M Neale; Baile, Clifton A; Murata, Tomiyasu

    2012-10-01

    Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss and hyperlipidemia. Consistent with these effects exogenous regucalcin has been shown to promote osteoclastogenesis in mouse bone marrow cultures and to suppress the differentiation and mineralization of MC3T3 osteoblast precursors. Regucalcin may induce hyperlipidemia in vivo by suppressing osteoblast differentiation and stimulating adipogenesis in bone marrow mesenchymal stem cells. The present study demonstrates that exogenous regucalcin suppresses differentiation to osteoblasts and stimulates adipogenesis in mouse bone marrow cell culture ex vivo. Moreover, exogenous regucalcin was found to enhance adipogenesis stimulated by insulin which is involved in the extracellular signal-related kinase pathway in 3T3-L1 adipocytes in vitro. PMID:22868942

  18. Characterization of in vitro cultured bone marrow and adipose tissue-derived mesenchymal stem cells and their ability to express neurotrophic factors.

    PubMed

    Taghi, Ghorbanian Mohammad; Ghasem Kashani Maryam, Haji; Taghi, Lashkarbolouki; Leili, Hosseinpour; Leyla, Mirzaeiyan

    2012-01-01

    MSCs (mesenchymal stem cells) have attracted attention as a promising tool for regenerative medicine and transplantation therapy. MSCs exert neuroprotective effects by secreting a number of factors in vitro and in vivo. Similar characteristics are found in ADSCs (adipose-derived stem cells) and BMSCs (bone marrow stromal cells). Multipotent capability, easy accessibility and rapid proliferation of ADSCs have been established. Our main objective was to compare cell viability, growth rate, expression of neurotrophic factors and nestin genes in ADSCs and BMSCs. Cell doubling time and proliferation rate indicate that ADSCs has a higher proliferation rate than BMSCs. ADSCs and BMSCs express a similar pattern of CD71 and CD90 markers. Nestin immunostaining showed that ADSCs and BMSCs are immunopositive. The expression of neurotrophic factors genes in ADSCs proved similar to that of BMSCs genes. Thus adipose tissue stem cells with a high proliferation rate can express nestin and neurotrophic factor genes. Therefore ADSCs may be useful in future cell replacement therapies and help improve neurodegenerative diseases. PMID:22994924

  19. Development of osteogenic cell sheets for bone tissue engineering applications.

    PubMed

    Pirraco, Rogério P; Obokata, Haruko; Iwata, Takanori; Marques, Alexandra P; Tsuneda, Satoshi; Yamato, Masayuki; Reis, Rui L; Okano, Teruo

    2011-06-01

    The use of scaffolds in combination with osteogenic cells has been the gold standard in bone tissue engineering strategies. These strategies have, however, in many cases failed to produce the desired results due to issues such as the immunogenicity of the biomaterials used and cell necrosis at the bulk of the scaffold related to deficient oxygen and nutrients diffusion. Here, we originally propose the use of cell sheet (CS) engineering as a possible way to overcome some of these obstacles. Osteogenic CSs were fabricated by culturing rat bone marrow stromal cells in thermoresponsive culture dishes. The CSs were recovered from the dishes using a low-temperature treatment and then were implanted subcutaneously in nude mice. New bone formation was verified from day 7 post-transplantation using X-ray, microcomputed tomography, and histological analysis. The presence of a vascularized marrow was also verified in the newly formed bone after 6 weeks of transplantation. Further, osteocytes were found in this newly formed tissue, supporting the conclusion that mature bone was formed after ectopically transplanting osteogenic CSs. These results therefore confirm the great potentiality of CS engineering to be used in bone tissue engineering applications. PMID:21275832

  20. Stem cell plasticity in muscle and bone marrow.

    PubMed

    Goodell, M A; Jackson, K A; Majka, S M; Mi, T; Wang, H; Pocius, J; Hartley, C J; Majesky, M W; Entman, M L; Michael, L H; Hirschi, K K

    2001-06-01

    Recent discoveries have demonstrated the extraordinary plasticity of tissue-derived stem cells, raising fundamental questions about cell lineage relationships and suggesting the potential for novel cell-based therapies. We have examined this phenomenon in a potential reciprocal relationship between stem cells derived from the skeletal muscle and from the bone marrow. We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5 day in vitro culture were harvested and introduced into each of six lethally irradiated recipients together with distinguishable whole bone marrow cells. Six and twelve weeks later, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56%, indicating that the cultured muscle cells generated approximately 10- to 14-fold more hematopoietic activity than whole bone marrow. Although the identity of the muscle-derived hematopoietic stem cells is still unknown, they may be identical to muscle satellite cells, some of which lack myogenic regulators and could respond to hematopoietic signals. We have also found that stem cells in the bone marrow can contribute to cardiac muscle repair and neovascularization after ischemic injury. We transplanted highly purified bone marrow stem cells into lethally irradiated mice that subsequently were rendered ischemic by coronary artery occlusion and reperfusion. The engrafted stem cells or their progeny differentiated into cardiomyocytes and endothelial cells and contributed to the formation of functional tissue. PMID:11458510

  1. 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. PMID:24687524

  2. Aging, inflammation, stem cells, and bone healing.

    PubMed

    Gibon, Emmanuel; Lu, Laura; Goodman, Stuart B

    2016-01-01

    Complex interactions among cells of the monocyte-macrophage-osteoclast lineage and the mesenchymal stem cell-osteoblast lineage play a major role in the pathophysiology of bone healing. Whereas the former lineage directs inflammatory events and bone resorption, the latter represents a source of cells for bone regeneration and immune modulation. Both of these lineages are affected by increasing age, which is associated with higher baseline levels of inflammatory mediators, and a significant reduction in osteogenic capabilities. Given the above, fracture healing, osteoporosis, and other related events in the elderly present numerous challenges, which potentially could be aided by new therapeutic approaches to modulate both inflammation and bone regeneration. PMID:27006071

  3. Osteocytes: The master cells in bone remodelling.

    PubMed

    Prideaux, Matthew; Findlay, David M; Atkins, Gerald J

    2016-06-01

    Bone remodelling is an essential process for shaping and maintaining bone mass in the mature skeleton. During our lifetime bone is constantly being removed by osteoclasts and new bone is formed by osteoblasts. The activities of osteoclasts and osteoblasts must be regulated under a strict balance to ensure that bone homeostasis is maintained. Osteocytes, which form an extensive, multi-functional syncytium throughout the bone, are increasingly considered to be the cells that maintain this balance. Current research is elucidating key signalling pathways by which the osteocyte exerts control over the other cell types in bone and over its own activities, and potential ways in which these pathways may be exploited therapeutically. PMID:26927500

  4. Bone regeneration at dental implant sites with suspended stem cells.

    PubMed

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

    2014-10-01

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

  5. Chemosensitizing AML cells by targeting bone marrow endothelial cells.

    PubMed

    Bosse, Raphael C; Wasserstrom, Briana; Meacham, Amy; Wise, Elizabeth; Drusbosky, Leylah; Walter, Glenn A; Chaplin, David J; Siemann, Dietmar W; Purich, Daniel L; Cogle, Christopher R

    2016-05-01

    Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML. PMID:26898708

  6. Bone marrow (stem cell) donation

    MedlinePlus

    ... Discomfort from needles in the arms Bone marrow harvest. This minor surgery is done under general anesthesia. ... takes about an hour. After a bone marrow harvest, the donor stays in the hospital until he ...

  7. 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.

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

    PubMed

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

    2008-02-01

    Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-beta 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH. PMID:18252948

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

    PubMed

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

    2010-12-15

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

  10. Osteogenic potential of mandibular vs. long-bone marrow stromal cells.

    PubMed

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

    2010-11-01

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

  11. Bone repair using periodontal ligament progenitor cell-seeded constructs.

    PubMed

    Tour, G; Wendel, M; Moll, G; Tcacencu, I

    2012-08-01

    The success of tissue-engineering therapies is dependent on the ability of scaffolds to guide differentiation of progenitor cells. Here we present a new approach using a biomimetic construct composed of hydroxyapatite modified with an in vitro-derived extracellular matrix (HA-ECM) and seeded with periodontal ligament progenitor cells (PDLCs). The study aimed to investigate the effect of HA-ECM on osteogenic differentiation of PDLCs and in vivo evaluation of the PDLC-seeded HA-ECM constructs using a rat calvarial critical-sized defect model. After flow-cytometric phenotyping of PDLCs for typical mesenchymal stem cell markers, the PDLCs were cultured on HA-ECM or HA alone in osteogenic media and assessed by MTT, alkaline phosphatase (ALP) assays, and real-time qPCR at different time intervals after seeding. New bone formation induced by PDLC-seeded constructs was assessed by histomorphometric analysis at 12 weeks post-operatively. The PDLCs seeded on HA-ECM showed significantly higher ALP activity and up-regulation of bone-related genes. The treatment with PDLC-seeded HA-ECM significantly improved calvarial bone repair, with the highest amount of newly formed bone elicited by cell-seeded constructs cultured for 14 days. Our results highlight the PDLC-seeded HA-ECM constructs as a promising tool for craniofacial bone regeneration. PMID:22736447

  12. 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

  13. Effect of the surface topography of electrospun poly(ε-caprolactone)/poly(3-hydroxybuterate-co-3-hydroxyvalerate) fibrous substrates on cultured bone cell behavior.

    PubMed

    K-hasuwan, Prae-ravee; Pavasant, Prasit; Supaphol, Pitt

    2011-09-01

    The use of electrospun fibrous matrices as substrates for cell/tissue culture has usually been confined to those consisting of smooth fibers. Here, we demonstrated that in vitro responses of mouse-calvaria-derived preosteoblastic cells (MC3T3-E1) that had been cultured on electrospun fibrous substrates made from blend solutions of 50/50 w/w poly(ε-caprolactone) (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) of varying concentrations, ranging from 4 to 14 wt %, depended strongly on the topography of the individual fibers. As the concentration of the blend solutions increased from 4 to 14 wt %, the topography of the individual fibers changed from discrete beads/smooth fibers to beaded fibers/smooth fibers and finally to smooth fibers, and the average diameter of the individual, smooth fibers increased from ∼0.4 to ∼1.8 μm. The results clearly showed that MC3T3-E1 preferred the smooth hydrophilic surface of the fibrous substrate from 10 wt % PCL/PHBV solution because the cells appeared to attach, proliferate, and differentiate on the surface of this substrate particularly well. PMID:21790199

  14. Improved angiogenic potency by implantation of ex vivo hypoxia prestimulated bone marrow cells in rats.

    PubMed

    Li, Tao-Sheng; Hamano, Kimikazu; Suzuki, Kazuhiko; Ito, Hiroshi; Zempo, Nobuya; Matsuzaki, Masunori

    2002-08-01

    Therapeutic angiogenesis can be induced by local implantation of bone marrow cells. We tried to enhance the angiogenic potential of this treatment by ex vivo hypoxia stimulation of bone marrow cells before implantation. Bone marrow cells were collected and cultured at 33 degrees C under 2% O(2)-5% CO(2)-90% N(2) (hypoxia) or 95% air-5% CO(2) (normoxia). Cells were also injected into the ischemic hindlimb of rats after 24 h of culture. Hypoxia culture increased the mRNA expression of vascular endothelial growth factor (VEGF), vascular endothelial (VE)-cadherin, and fetal liver kinase-1 (Flk-1) from 2.5- to fivefold in bone marrow cells. The levels of VEGF protein in the ischemic hindlimb were significantly higher 1 and 3 days after implantation with hypoxia-cultured cells than with normoxia-cultured or noncultured cells. The microvessel density and blood flow rate in the ischemic hindlimbs were also significantly (P < 0.001) higher 2 wk after implantation with hypoxia-cultured cells (89.7 +/- 5.5%) than with normoxia-cultured cells (67.0 +/- 9.6%) or noncultured cells (70.4 +/- 7.7%). Ex vivo hypoxia stimulation increased the VEGF mRNA expression and endothelial differentiation of bone marrow cells, which together contributed to improved therapeutic angiogenesis in the ischemic hindlimb after implantation. PMID:12124190

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

    PubMed

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

    2003-11-01

    In the traditional Korean medicine, Drynariae Rhizoma (DR) [Drynaria fortunei (kunze) J. Sm] has been reported as a good enhancer for bone healing. In this experiment, we investigate the effects of DR on bone resorption using the bone cells culture. Different concentrations of crude extract of DR were added to mouse bone cells culture. The mitochondria activity of the bone cells after exposure was determined by colorimetric MTT assay. It was demonstrated that DR has potential effects on the bone cells culture without any cytotoxicity. The most effective concentration of DR on bone cells was 100 micro g/ml. On the other hand, cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. In this study, Mouse long bone cells including osteoclasts and osteoblast were treated with the PI3-kinase inhibitor, wortmannin (WT), and a specific inhibitor of protein kinase C (PKC), calphostin C. Although WT prevented the osteoclast-mediated intracellular processing of Cat K, calphostin C did not. Similarly, treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extracts prevented the intracellular maturation of Cat K, suggesting that DR may disrupt the intracellular trafficking of pro Cat K. This is similar to that of WT. Since secreted proenzymes have the potential to reenter the cell via mannose-6-phosphate (M6P) receptor, to prevent this possibility, we tested WT and DR in the absence or presence of M6P. Inhibition of Cat K processing by WT or DR was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of WT and DR. DR dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing. PMID:14555293

  16. 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. PMID:23642054

  17. 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

  18. Fish oil prevents breast cancer cell metastasis to bone.

    PubMed

    Mandal, Chandi Charan; Ghosh-Choudhury, Triparna; Yoneda, Toshi; Choudhury, Goutam Ghosh; Ghosh-Choudhury, Nandini

    2010-11-26

    The data derived from epidemiological and animal models confirm a beneficial effect of fish oil (rich in ω-3 polyunsaturated fatty acids) in the amelioration of tumor growth and progression, including breast cancer. The breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Using a mouse model of MDA-MB-231 human breast cancer cell metastasis to bone, here we show that fish oil diet enriched in DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) prevents the formation of osteolytic lesions in bone, indicating suppression of cancer cell metastasis to bone. These results are supported by our data showing both DHA and EPA significantly attenuate the migration/invasion of MDA-MB-231 breast cancer cells in culture. The mechanism that limits breast cancer cells to selective metastasis to bone remains hitherto unexplored. Aberrant increased expression of CD44 is associated with generation of cancer stem cells, which contribute to metastasis of breast cancer cells. We demonstrate that DHA and EPA significantly inhibit the expression of CD44 protein and mRNA by a transcriptional mechanism. Furthermore, we show markedly reduced levels of CD44 mRNA and protein in the tumors of mice, which were fed fish oil diet than those in control diet. Our data provide the first evidence for a salutary effect of fish oil on breast cancer metastasis to bone. Our results identify a novel function of the fish oil active components, DHA and EPA, which target the cell-intrinsic pro-metastatic molecule CD44 to inhibit migration/invasion. PMID:20971068

  19. Stem Cells in Teeth and Craniofacial Bones.

    PubMed

    Zhao, H; Chai, Y

    2015-11-01

    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

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

    PubMed Central

    Crane, Janet L.; Cao, Xu

    2014-01-01

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

  1. [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(CD31hiEmcnhi, type H), while the endothelial cells of sinusoidal vessels in diaphysis revealed only weak CD31/Endomucin(CD31loEmcnlo, type L).It is suggested crucial roles of endothelial HIF in controlling bone angiogenesis, type H vessel abundance, endothelial growth factor expression and osteogenesis. PMID:27117612

  2. Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture.

    PubMed Central

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

    1987-01-01

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

  3. Stem cells and bone: a historical perspective.

    PubMed

    Bianco, Paolo

    2015-01-01

    Bone physiology and stem cells were tightly intertwined with one another, both conceptually and experimentally, long before the current explosion of interest in stem cells and so-called regenerative medicine. Bone is home to the two best known and best characterized systems of postnatal stem cells, and it is the only organ in which two stem cells and their dependent lineages coordinate the overall adaptive responses of two major physiological systems. All along, the nature and the evolutionary significance of the interplay of bone and hematopoiesis have remained a major scientific challenge, but also allowed for some of the most spectacular developments in cell biology-based medicine, such as hematopoietic stem cell transplantation. This question recurs in novel forms at multiple turning points over time: today, it finds in the biology of the "niche" its popular phrasing. Entirely new avenues of investigation emerge as a new view of bone in physiology and medicine is progressively established. Looking at bone and stem cells in a historical perspective provides a unique case study to highlight the general evolution of science in biomedicine since the end of World War II to the present day. A paradigm shift in science and in its relation to society and policies occurred in the second half of the XXth century, with major implications thereof for health, industry, drug development, market and society. Current interest in stem cells in bone as in other fields is intertwined with that shift. New opportunities and also new challenges arise. This article is part of a Special Issue entitled "Stem cells and bone". PMID:25171959

  4. 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

  5. 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.

  6. 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

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

    PubMed

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

    2014-06-27

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

  8. Effect of a titanium surface on bone marrow-derived osteoblastic cells in vitro.

    PubMed

    Kurachi, T; Nagao, H; Nagura, H; Enomoto, S

    1997-06-01

    The initial interaction between a titanium implant and the surrounding cancellous bone tissue was investigated by means of in vitro cultures of bone marrow-derived osteoblastic clone TMS-12 cells. Proliferation of TMS-12 cells cultured on a titanium surface was significantly reduced compared with that of control cells cultured directly on plastic, but alkaline phosphatase activity was the same as control cells. Co-culture of spleen cells with TMS-12 cells in the presence of a 1 alpha,25-dihydroxyvitamin D3 on titanium resulted in significant inhibition of tartrate-resistant acid phosphatase activity compared to control cultures on plastic. More prostaglandin E2 (PGE2) was produced by cells grown on a titanium surface than on plastic tissue-culture plates. On the other hand, mouse calvaria-derived osteogenic MC3T3-E1 cells, known to proliferate well on titanium, released similar amounts of PGE2 on titanium surfaces as on plastic tissue culture plates, indicating additional marked differences between the two osteoblastic cell types in response to a titanium surface. These results suggest that the reaction of bone marrow-derived osteoblastic cells to a titanium surface may fundamentally differ from that of calvaria-derived osteoblastics even though both cells are from bone tissue. PMID:9382711

  9. 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.

  10. Concise Review: Cell-Based Strategies in Bone Tissue Engineering and Regenerative Medicine

    PubMed Central

    Ma, Jinling; Both, Sanne K.; Yang, Fang; Cui, Fu-Zhai; Pan, Juli; Meijer, Gert J.; Jansen, John A.

    2014-01-01

    Cellular strategies play an important role in bone tissue engineering and regenerative medicine (BTE/RM). Variability in cell culture procedures (e.g., cell types, cell isolation and expansion, cell seeding methods, and preculture conditions before in vivo implantation) may influence experimental outcome. Meanwhile, outcomes from initial clinical trials are far behind those of animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the BTE/RM constructs as some complex clinical implementations require bone regeneration in too large a quantity. Coculture strategies, in which angiogenic cells are introduced into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, preclinical studies have demonstrated that cell-based tissue-engineered constructs generally induce more bone formation compared with acellular constructs. Further, cocultures have been shown to enhance vascularization and bone formation compared with monocultures. However, translational efficacy from animal studies to clinical use requires improvement, and the role implanted cells play in clinical bone regeneration needs to be further elucidated. In view of this, the present review provides an overview of the critical procedures during in vitro and in vivo phases for cell-based strategies (both monoculture and coculture) in BTE/RM to achieve more standardized culture conditions for future studies, and hence enhance bone formation. PMID:24300556

  11. Bone marrow mesenchymal stem cells: historical overview and concepts

    PubMed Central

    Charbord, Pierre

    2010-01-01

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

  12. 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

  13. Huanglongbing and psyllid cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Production scale insect cell culture.

    PubMed

    Agathos, S N

    1991-01-01

    Insect cells in culture are currently commanding great interest as superior hosts for the efficient production of biologicals with applications in health care and in agriculture. Insect cell culture is ripe for scale-up technologies, in order to meet future projected production requirements of (a) insect viruses used as bioinsecticides and (b) recombinant proteins of therapeutic potential for humans and animals. The single most prominent system used in research-based and in commercial insect cell culture today involves lepidopteran cells transfected with baculovirus expression vectors for abundant formation of recombinant biologicals. However, dipteran insect cell lines also are beginning to emerge as useful tools in biotechnology. Current practices in bioprocess development using insect cell culture, advances in media formulation and in insect cell bioreactor design, and emerging trends are presented and critically evaluated. PMID:14543739

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  17. 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.

  18. Putative human male germ cells from bone marrow stem cells.

    PubMed

    Drusenheimer, Nadja; Wulf, Gerald; Nolte, Jessica; Lee, Jae Ho; Dev, Arvind; Dressel, Ralf; Gromoll, Jörg; Schmidtke, Jörg; Engel, Wolfgang; Nayernia, Karim

    2007-01-01

    Germ cells must develop along distinct male or female paths to produce the spermatozoa or oocyte required for sexual reproduction. Male germline stem cells maintain spermatogenesis in the postnatal human testis. Here we show that a small population of bone marrow cells is able to transdifferentiate to male germ cell-like cells. We show expression of early germ cell markers (Oct4, Fragilis, Stella and Vasa) and male germ cell specific markers (Dazl, TSPY, Piwil2 and Stra8) in these cells. Our preliminary findings provide direct evidence that human bone marrow cells can differentiate to putative male germ cells and identify bone marrow as a potential source of male germ cells that could sustain sperm production. PMID:17566262

  19. Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

    PubMed

    Kitami, Megumi; Kaku, Masaru; Rocabado, Juan Marcelo Rosales; Ida, Takako; Akiba, Nami; Uoshima, Katsumi

    2016-09-01

    Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc. PMID:26754153

  20. 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

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

    SciTech Connect

    Cowan, Robert W.; Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 ; Ghert, Michelle; Department of Surgery, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 ; Singh, Gurmit; Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2

    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 results suggest that T cells may potentiate the catabolic effect of GCT.

  3. 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.

  4. 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. PMID:18847330

  5. Comparison of endometrial regenerative cells and bone marrow stromal cells

    PubMed Central

    2012-01-01

    Background Endometrial regenerative cells (ERC) and bone marrow stromal cells (BMSC) are being used in clinical trials. While they have been reported to have similar characteristics, they have not been directly compared. Methods We compared micro RNA (miRNA) and gene expression profiles, soluble cytokine and growth factor levels and ability to inhibit ongoing mixed leukocyte reaction (MLR) of ERC and BMSC each derived from 6 healthy subjects. Results ERC and BMSC miRNA and gene expression profiles were similar, but not identical; more differences were noted in the expression of genes than in miRNAs. Genes overexpressed in ERCs were more likely to be in immune and inflammation pathways and those overexpressed in BMSCs were more likely to be in stem cell and cancer signaling pathways. In addition, the levels of IL-8 and ICAM-1 were greater in ERC supernatants while the levels of HGF, VEGF, IL-6, CXCL12, TGFB1 and TGFB2 were greater in BMSC supernatants. Additionally, ERC demonstrated greater inhibition of the proliferation of mixed leukocyte cultures. Conclusions These results suggest that the in vivo effects of ERC and BMSC may differ. Multiple properties of stromal cells are responsible for their in vivo effectiveness and ERC may be more effective for some of the clinical applications and BMSC for others. Studies in animal models or clinical trials will be required to more fully characterize the differences between ERC and BMSC. PMID:23038994

  6. Three-dimensional culture may promote cell reprogramming

    PubMed Central

    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

  7. Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone.

    PubMed

    Wang, Ruoxiang; Xu, Jianchun; Juliette, Lisa; Castilleja, Agapito; Love, John; Sung, Shian-Ying; Zhau, Haiyen E; Goodwin, Thomas J; Chung, Leland W K

    2005-10-01

    Cancer-stromal interaction results in the co-evolution of both the cancer cells and the surrounding host stromal cells. As a consequence of this interaction, cancer cells acquire increased malignant potential and stromal cells become more inductive. In this review we suggest that cancer-stromal interaction can best be investigated by three-dimensional (3D) co-culture models with the results validated by clinical specimens. We showed that 3D culture promoted bone formation in vitro, and explored for the first time, with the help of the astronauts of the Space Shuttle Columbia, the co-culture of human prostate cancer and bone cells to further understand the interactions between these cells. Continued exploration of cancer growth under 3D conditions will rapidly lead to new discoveries and ultimately to improvements in the treatment of men with hormonal refractory prostate cancer. PMID:15982899

  8. Maintenance of hematopoiesis in serum-free bone marrow cultures involves sequential recruitment of quiescent progenitors.

    PubMed

    Lansdorp, P M; Dragowska, W

    1993-09-01

    We previously described that cells with a CD34+CD71lo phenotype from adult human bone marrow are maintained at constant numbers in long-term suspension cultures supplemented with interleukin-6 (IL-6), IL-3, mast growth factor (MGF) (a c-kit ligand), and erythropoietin (Epo). In view of the large increase in cell numbers in such cultures (for example, > 10(6)-fold per cell), this was an unexpected finding. The following models for the observed maintenance of CD34+CD71lo cells in our cultures were considered: (1) survival of non-dividing cells; (2) self-renewal balanced by loss of cells; (3) asymmetrical divisions; and (4) combinations of the above. Two experimental strategies were explored to discriminate between these models. In the first, sorted CD34+CD45RAloCD71lo cells were labeled with the flourescent tracking dye PKH26, followed by analysis of PKH26 fluorescence of CD34+CD71lo and other cells present in the cultures at various times (up to 11 weeks). In the second approach, single CD34+CD45RAloCD71lo cells were directly sorted into individual wells, and growing cells were then analyzed by flow cytometry. Results from these experiments indicated a considerable variability in (1) the number of surviving input cells (ranging from 30 to 80%); (2) the proportion of cells that contributed significantly to the total cell production measured at day 20 (ranging from 1 to 5%); and (3) the number of CD34+ cells present in individual clones. Taken together, the observed maintenance of primitive CD34+ cells in our cultures apparently involves a combination of survival of CD34+CD71lo cells with a vary low turnover together with a very limited production of CD34+ cells. Clonal heterogeneity, differences in cell cycle kinetics between CD34+ and CD34- cells, and observations that the majority of bone marrow-derived CD34+CD45RAloCD71lo cells do not show a rapid proliferative response to a mixture of IL-6, IL-3, MGF, and Epo will have to be taken into account in the development of experimental strategies aimed at clinically useful expansion of primitive hematopoietic cells ex vivo. PMID:7689481

  9. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells

    PubMed Central

    Wu, Yuxin; Zhang, Jinghan; Ben, Xiaoming

    2013-01-01

    Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the “pour-off” method. Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts, and could be expanded by supplementation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo. PMID:25206516

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

    PubMed Central

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

    1991-01-01

    To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis. Images PMID:1845868

  11. 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.

  12. Cell culture purity issues and DFAT cells

    SciTech Connect

    Wei, Shengjuan; Department of Animal Sciences, Washington State University, Pullman, WA 99164 ; 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.

  13. A compact, automated cell culture system for clinical scale cell expansion from primary tissues.

    PubMed

    Kato, Ryuji; Iejima, Daisuke; Agata, Hideki; Asahina, Izumi; Okada, Kunihiko; Ueda, Minoru; Honda, Hiroyuki; Kagami, Hideaki

    2010-10-01

    Despite the growing number of clinically practical automated cell culture systems, demand is also increasing for more compact platforms with greater capabilities to prepare primary cells directly from patient tissue. Here we report the development of an automated cell culture system that is also compact. The machinery consisted of a supply unit, an incubation unit, and a collection unit, which fit within a 70 cm x 60 cm x 86 cm space. The compact size was enabled by our concept of using a single culture vessel from the primary culture steps to final cell harvest instead of scaling up with multiple culture vessels. Human fibroblasts and bone marrow stromal cells (BMSCs) were successfully cultured with this system over 19 days without contamination. From three pieces of gingival tissue (2 mm x 2 mm) or from 10 mL of bone marrow aspirate, the system could produce more than 2.0x10(7) cells and up to 3.0x10(7) cells for fibroblasts and BMSCs, respectively. The BMSCs produced by this system were capable of ectopic bone formation after transplantation into the subcutaneous space of nude mice. Our prototype system will provide a foundation for minimizing automatic culture machinery with clinically relevant cell yields while also expanding the automation capabilities to include primary tissue culture. PMID:19958165

  14. Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells.

    PubMed

    Kim, Hyeon Joo; Kim, Ung-Jin; Vunjak-Novakovic, Gordana; Min, Byoung-Hyun; Kaplan, David L

    2005-07-01

    The aim of this study was to investigate the effect of three-dimensional silk fibroin scaffold preparation methods (aqueous and solvent) on osteogenic responses by human bone marrow stem cells (hMSCs). Macroporous 3D protein scaffolds with similar sized pores of 900+/-50 microm were prepared either by an organic solvent process (hexafluoro-2-propanol, HFIP) or an aqueous process. hMSCs were expanded, seeded on the scaffolds, and cultured up to 28 days under static conditions in osteogenic media. hMSCs seeded onto the water-based silk scaffolds showed a significant increase in cell numbers (p<0.01) vs. the HFIP-prepared silk scaffolds. Significantly higher (p<0.01) alkaline phosphatase (ALPase) activity and calcium deposition were apparent after 28 days of culture in the water-based silk scaffolds when compared to the HFIP-derived silk scaffolds. Transcript levels for collagen type I (Col I), ALP, and osteopontin (OP) increased (p<0.05) in the water-based silk scaffolds in comparison to the HFIP-derived materials. At early stages of culture, increased expression of OP and collagen type II (Col II) were also observed in both scaffolds. Expression of Col II, MMP 13, Col I, and OP proteins increased in the water-based silk scaffolds in comparison to the HFIP-derived scaffolds while bone sialoprotein (BSP) proteins increased in the HFIP-derived silk scaffolds in comparison to the water-based scaffolds after 28 days of culture. Histological analysis showed the development of bone-like trabeculae with cuboid cells in an extracellular matrix (ECM) in the water-based silk scaffolds with more organization than in the HFIP-derived material after 28 days of culture. Alcian blue staining demonstrated the presence of proteoglycan in the ECM formed in the water-based scaffolds but not in the HFIP-prepared silk scaffolds. The results suggest that macroporous 3D aqueous-derived silk fibroin scaffolds provide improved bone-related outcomes in comparison to the HFIP-derived systems. These data illustrate the importance of materials processing on biological outcomes, as the same protein, silk fibroin, was used in both preparations. PMID:15701373

  15. Scientists Find Stem Cells That Might Repair Skull, Face Bones

    MedlinePlus

    ... gov/medlineplus/news/fullstory_157041.html Scientists Find Stem Cells That Might Repair Skull, Face Bones Discovery made ... they may be one step closer to using stem cells to replace damaged skull and facial bones in ...

  16. Immunohistochemical characteristics of bone forming cells in pleomorphic adenoma.

    PubMed

    Nakano, Keisuke; Watanabe, Takehiro; Shimizu, Takako; Kawakami, Toshiyuki

    2007-01-01

    Histopathological and immunohistochemical examinations were carried out in a case of pleomorphic adenoma with bone formation, occurring in the chin of a 34-year-old Japanese man. Examination results showed the modified neoplastic myoepithelial cells reacted positively to S-100 protein. The S-100-positive modified neoplastic myoepithelial cells were proliferated in the closely related area of the bone tissue. Furthermore, positive reaction was detected in the bone forming cells: osteoblasts and osteocytes. These cells also reacted positively to Runx2 as a marker of bone forming cells. These results suggest that the origin of the bone forming cells in this case of pleomorphic adenoma was modified neoplastic myoepithelial cells. PMID:18026564

  17. 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. PMID:21312336

  18. 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.

  19. Differential marker expression by cultures rich in mesenchymal stem cells

    PubMed Central

    2013-01-01

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

  20. 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.

  1. 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 mammary fat pads of mice not implanted with human bone discs. PMID:26231669

  2. 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

  3. Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    PubMed Central

    Zhang, Pei-xun; Jiang, Xiao-rui; Wang, Lei; Chen, Fang-min; Xu, Lin; Huang, Fei

    2015-01-01

    Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons (sensory neurons) from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteogenic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the proliferation of bone marrow mesenchymal stem cell-derived osteoblasts at 3 and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-related factors (including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2) in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which provides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone. PMID:25788931

  4. Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture.

    PubMed

    Gulati, Twishi; Chung, Sylvia A; Wei, Ai-Qun; Diwan, Ashish D

    2015-12-01

    Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p<0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells. PMID:26134557

  5. 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

  6. 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.

  7. Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells.

    PubMed

    Ezeh, Peace C; Xu, Huan; Wang, Shu Chun; Medina, Sebastian; Burchiel, Scott W

    2016-01-01

    Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM microenvironment. Toxicological investigations following in vivo exposures of mice or in vitro exposures of mouse primary BM cell cultures allow the assessment of the developmental and functional integrity of BM cells, cell population shifts, and adverse biochemical effects due to toxicity. Colony forming unit (CFU) assays and flow cytometry are indispensable techniques in these toxicity studies. © 2016 by John Wiley & Sons, Inc. PMID:26828331

  8. 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

  9. Bone Repair Cells for Craniofacial Regeneration

    PubMed Central

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

    2012-01-01

    Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both Somatic and Stem Cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice. PMID:22433781

  10. BMP4 can generate primordial germ cells from bone-marrow-derived pluripotent stem cells.

    PubMed

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

    2012-01-01

    Evidence of germ cell derivation from embryonic and somatic stem cells provides an in vitro model for the study of germ cell development, associated epigenetic modification and mammalian gametogenesis. More importantly, in vitro derived gametes also represent a potential strategy for treating infertility. In mammals, male and female gametes, oocyte and sperm, are derived from a specific cell population, PGCs (primordial germ cells) that segregate early in embryogenesis. We have isolated pluripotent SSEA-1+ (stage-specific embryonic antigen-1+) cells from mice bone marrow using a MACS (magnetic-activated cell sorting) system. SSEA-1+ cells were directly separated from the suspension of MMCs (murine mononuclear cells) harvested from bone marrow of 2-4-week-old mice. Flow-cytometry assay immediately after sorting and culturing under undifferentiated condition showed 55±7% and 87±4% purity respectively. RT-PCR (reverse transcription-PCR) analysis after differentiation of SSEA-1+ cells into derivations of three germ layers showed the pluripotency properties of isolated cells. SSEA-1+ cells were induced to differentiate along germ cell lineage by adding BMP4 (bone morphogenic factor-4) to the medium. Regarding the expression of germ cell markers (PGCs, male and female germ cell lineage), it was found that adding exogenous BMP4 to culture medium could differentiate pluripotent SSEA-1+ cells isolated from an adult tissue into gamete precursors, PGCs. Differentiated cells expressed specific molecular markers of PGCs, including Oct4, fragilis, Stella and Mvh (mouse vasa homologue). Therefore BMP4 is insufficient to induce SSEA-1+ cells derived from PGCs to develop further into late germ cells in vitro. PMID:22988836

  11. 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.

  12. Dissecting the Role of Bone Marrow Stromal Cells on Bone Metastases

    PubMed Central

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

    2014-01-01

    Tumor-induced bone disease is a dynamic process that involves interactions with many cell types. Once metastatic cancer cells reach the bone, they are in contact with many different cell types that are present in the cell-rich bone marrow. These cells include the immune cells, myeloid cells, fibroblasts, osteoblasts, osteoclasts, and mesenchymal stem cells. Each of these cell populations can influence the behavior or gene expression of both the tumor cells and the bone microenvironment. Additionally, the tumor itself can alter the behavior of these bone marrow cells which further alters both the microenvironment and the tumor cells. While many groups focus on studying these interactions, much remains unknown. A better understanding of the interactions between the tumor cells and the bone microenvironment will improve our knowledge on how tumors establish in bone and may lead to improvements in diagnosing and treating bone metastases. This review details our current knowledge on the interactions between tumor cells that reside in bone and their microenvironment. PMID:25054153

  13. CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction.

    PubMed

    Oue, Erika; Lee, Ji-Won; Sakamoto, Kei; Iimura, Tadahiro; Aoki, Kazuhiro; Kayamori, Kou; Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo; Yamaguchi, Akira

    2012-08-01

    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 report showing the role of CXCL2 in cancer-associated bone destruction. PMID:22771802

  14. Value of surveillance cultures in a bone marrow transplantation unit.

    PubMed

    Czirók, E; Prinz, G Y; Dénes, R; Reményi, P; Herendi, A

    1997-09-01

    Because of the increased risk of infection with the associated diagnostic and therapeutic problems in bone marrow transplantation (BMT) patients, the usefulness of surveillance cultures (SC) at the BMT department of the National Institute of Haematology, Blood Transfusion, Transplantation and Immunology, Budapest, was reviewed. Between January 1992 and May 1995, 26 BMT operations were performed; 13 patients had 23 febrile espisodes. In 12 of these episodes infection was clinically documented; however, SC of these patients yielded bacteria identical with those in the blood culture in only two episodes (1 and 6 days before their blood cultures became positive, respectively). Out of a total of 1187 samples from these patients, potentially pathogenic bacteria were isolated from 145 SC and 43 blood cultures (drawn on 31 different days). Suppression of the gastrointestinal flora could be achieved by the department's decontamination regimen; however, overgrowth by gram-positive organisms (mainly coagulase-negative staphylococci) occurred in the intestine and at other body sites. On the basis of these results, SC are of limited value in predicting infection or identifying the causative organisms of fever. On the other hand, SC are useful in confirming the efficiency of suppression of the body flora by antimicrobial agents. Specific treatment was based on suitably sampled materials, and close contact between physicians, infectious disease specialists and microbiologists was essential. PMID:9291891

  15. 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

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

    PubMed

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

    2012-04-01

    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

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

    TOXLINE Toxicology Bibliographic Information

    Eick JD; Barragan-Adjemian C; Rosser J; Melander JR; Dusevich V; Weiler RA; Miller BD; Kilway KV; Dallas MR; Bi L; Nalvarte EL; Bonewald LF

    2012-04-01

    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.

  18. 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

  19. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

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

  20. Recent advances in bone regeneration using adult stem cells

    PubMed Central

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

    2015-01-01

    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. PMID:25914769

  1. 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. PMID:25914769

  2. 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.

  3. 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.

  4. Participation of bone marrow derived cells in cutaneous wound healing.

    PubMed

    Badiavas, Evangelos V; Abedi, Mehrdad; Butmarc, Janet; Falanga, Vincent; Quesenberry, Peter

    2003-08-01

    Bone marrow has long been known to be a source of stem cells capable of regeneration of the hematopoeitic system. Recent reports, however, have indicated that bone marrow might also contain early stem cells that can differentiate into other organ tissues such as skin. While these studies have illustrated that bone marrow stem cells could find their way to the skin, they have not addressed the dynamics of how bone marrow stem cells might participate in the homeostatis and regeneration of skin. In this report we followed green fluorescent protein (GFP) labeled bone marrow transplanted into non-GFP mice in order to determine the participation of bone marrow stem cells in cutaneous wounds. Our results indicate that there are a significant number of bone marrow cells that traffic through both wounded and non-wounded skin. Wounding stimulated the engraftment of bone marrow cells to the skin and induced bone marrow derived cells to incorporate into and differentiate into non-hematopoietic skin structures. This report thus illustrates that bone marrow might be a valuable source of stem cells for the skin and possibly other organs. Wounding could be a stimulus for bone marrow derived stem cells to travel to organs and aid in the regeneration of damaged tissue. PMID:12811816

  5. Principles of cancer cell culture.

    PubMed

    Cree, Ian A

    2011-01-01

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

  6. Derivation of male germ cell-like lineage from human fetal bone marrow stem cells.

    PubMed

    Hua, Jinlian; Pan, Shaohui; Yang, Chunrong; Dong, Wuzi; Dou, Zhongying; Sidhu, Kuldip S

    2009-07-01

    Mesenchymal stem cells derived from bone marrow are a well characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. Reports have shown that murine and human embryonic stem cells can differentiate into primordial germ cells and then to early gametes. Evidence has indicated that some adult stem cells also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of human mesenchymal stem cells into germ cells. This study investigated the ability of retinoic acid and testicular extracts to induce human bone marrow stem cells (hBMSC) to differentiate into male germ cells. It was found that a small population of hBMSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers OCT4, STELLA, NANOG and VASA, and male germ-ceil-specific markers such as DAZL, TH2, c-kit, beta(1)-integrin, ACR, PRMl, FSHR, STRA8 and SCP3, as analysed by reverse transcription-polymerase chain reaction and immunohistochemistry. These results demonstrated that hBMSC may differentiate into male germ cells and the same could be used as a potential source of cells for reproductive toxicological studies. PMID:19573297

  7. Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone.

    PubMed

    Uchihara, Yoshinobu; Akahane, Manabu; Shimizu, Takamasa; Ueha, Tomoyuki; Morita, Yusuke; Nakasaki, Shintaro; Kura, Tomohiko; Tohma, Yasuaki; Kido, Akira; Kawate, Kenji; Tanaka, Yasuhito

    2015-01-01

    Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors. PMID:26064933

  8. 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

  9. 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

  10. 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

  11. Langerhans Cell Histiocytosis of the Temporal Bone.

    PubMed

    Ginat, Daniel Thomas; Johnson, Daniel N; Cipriani, Nicole A

    2016-06-01

    Langerhans cell histiocytosis involving the temporal bone region is uncommon and can resemble malignant neoplasms on imaging due to high cellularity. Although recognizing the presence of sharp margins with beveled-edges can be helpful, tissue sampling is often necessary for confirming the diagnosis. Cytology classically demonstrates kidney-bean shaped nuclei within the Langerhans cells and immunohistochemical staining is positive for S-100, peanut agglutinin (PNA), MHC class II, CD1a, and Langerin (CD 207). These features are exemplified in this sine qua non radiology-pathology correlation article. PMID:25903273

  12. Migration of osteoblastic cells on various guided bone regeneration membranes.

    PubMed

    Takata, T; Wang, H L; Miyauchi, M

    2001-08-01

    To evaluate the biological effects of guided bone regeneration (GBR) barrier materials on osteoblastic cell migration, migration of mouse osteoprogenitor cells (MC3T3-E1) was examined, in vitro, on various membranes. Eight commercially available GBR membranes - bovine type I collagen (BioMend; BM), porcine type I collagen (BioGide; BG), bovine type I atelocollagen (Tissue Guide; TG), polylactic acid (Epi-Guide; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute; RL, Resolut XT; RL-XT), expanded polytetrafluoroethylene (e-PTFE; Gore Tex; GT) and co-polymer of cellulose acetate and nitrocellulose (Millipore filter; MP) - were tested. A 3x5 mm section of the membrane was fixed to the bottom of a culture dish with double-sided adhesive tape, and half of the membrane was closely covered by PARAFILM (American National Can) to leave an unexposed area for cell migration. The border between exposed and unexposed areas was marked as a baseline of cell migration. Membranes were then plated with 3 ml of cell suspension at an initial density of 1x105 cells/ml in alpha-MEM culture medium with 10% fetal bovine serum and ascorbic acid. After a 5-hour incubation, non-attached cells were completely washed out with phosphate buffered saline and the PARAFILM cover was removed. After 3 days cultivation, specimens were fixed with 10% buffered formalin and stained briefly with hematoxylin. The area of cell migration on a membrane was analyzed using a LA 500 Image Analysis System and migration area per unit length of the baseline (mm2/mm) was compared among membranes. Results demonstrated that cell migration was greater in the order: RL>RL-XT, BM, TG, MP>EG, BG. Membranes except for BG, EG and GT showed the migration rate equal to or higher than a plastic culture cover slip (Celldesk) (P<0.01) on which cells generally grow favorably. Only a small number of the cells attached to GT, and the net cell migration for the membrane could not be determined. These results indicate that GBR barrier materials per se may influence the process of bone regeneration in vivo through the effects of their presence on cell migration. PMID:11488862

  13. Connecting Mechanics and Bone Cell Activities in the Bone Remodeling Process: An Integrated Finite Element Modeling

    PubMed Central

    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 adaptation. The proposed FEM model gives insight into how bone cells adapt their architecture to the mechanical and biological environment. PMID:25152881

  14. 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 adaptation. The proposed FEM model gives insight into how bone cells adapt their architecture to the mechanical and biological environment. PMID:25152881

  15. 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

  16. Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures.

    PubMed

    Malaval, L; Gupta, A K; Aubin, J E

    1995-04-01

    Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with both anabolic and catabolic effects on bone tissue. To investigate the effect of LIF on bone formation in the absence of a resorption cycle, we used fetal rat calvaria cell cultures and quantified bone nodule production, which provides a colony assay to analyze the effects of factors on osteoprogenitor differentiation and bone formation. In these cultures, dexamethasone (Dex) stimulates bone nodule formation. In dose-response experiments, LIF inhibited bone nodule formation by cells cultured with (+Dex; ID50 = 250 U/ml) or without (-Dex; ID50 = 30 U/ml) 10(-8) M Dex. Residual nodules were small and poorly mineralized. Continuous exposure to LIF (500 U/ml) up to day 25 did not affect either the growth rate or saturation density of the cultures, but decreased alkaline phosphatase activity and bone nodule production, with greater inhibition in -Dex cultures. Exposure to LIF (500 U/ml) for 3 days early during nodule formation (about day 10) reduced bone nodule numbers to the same extent as continuous treatment in -Dex cultures and significantly, but less markedly, in +Dex cultures; earlier and later pulses had no effect. Northern blot analysis of expression of messenger RNAs of bone related proteins in cultures pulsed (-Dex) at various stages of development showed marked inhibition of alkaline phosphatase, bone sialoprotein, and osteocalcin; slight inhibition of type I collagen; early stimulation of osteopontin; and no effect on Secreted Protein, Acidic and Rich in Cysteine/osteonectin. These results suggest that LIF is an inhibitor of bone nodule formation in these cultures, acting at a stage when late osteoprogenitors and/or early osteoblasts are present, and that Dex may modulate the effects of LIF by shifting effective doses to higher concentrations. PMID:7895651

  17. Injectable Bone Tissue Engineering Using Expanded Mesenchymal Stem Cells

    PubMed Central

    Yamada, Yoichi; Nakamura, Sayaka; Ito, Kenji; Umemura, Eri; Hara, Kenji; Nagasaka, Tetsuro; Abe, Akihiro; Baba, Shunsuke; Furuichi, Yasushi; Izumi, Yuichi; Klein, Ophir D.; Wakabayashi, Toshihiko

    2014-01-01

    Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, very few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue-engineered bone composed of mesenchymal stem cells (MSCs) and platelet rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out pilot trial cases in patients with long-term follow up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months was significantly higher than the pre-operation baseline (P <0.001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long term follow-up. Injectable tissue-engineered bone restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering. PMID:23225744

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

    PubMed Central

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

    2014-01-01

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

  19. 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.

  20. 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. PMID:26530150

  1. 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. PMID:25918341

  2. 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

  3. 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 represent a paradigm shift describing the mechanism of endochondral bone repair and open the door for novel regenerative strategies based on improved biology. PMID:24259230

  4. 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.

  5. Mesenchymal stem cells from patients to assay bone graft substitutes.

    PubMed

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

    2013-06-01

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

  6. 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.

  7. Fibromodulin reprogrammed cells: A novel cell source for bone regeneration.

    PubMed

    Li, Chen-Shuang; Yang, Pu; Ting, Kang; Aghaloo, Tara; Lee, Soonchul; Zhang, Yulong; Khalilinejad, Kambiz; Murphy, Maxwell C; Pan, Hsin Chuan; Zhang, Xinli; Wu, Benjamin; Zhou, Yan-Heng; Zhao, Zhihe; Zheng, Zhong; Soo, Chia

    2016-03-01

    Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However, currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability, painful and invasive cell-harvesting procedures, and tumorigenesis. Previously, we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan, fibromodulin (FMOD), circumventing gene transduction. In this study, we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect, contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario invivo. The persistence, engraftment, and osteogenesis of transplanted FReP cells without tumorigenesis invivo were confirmed by histological and immunohistochemical staining. Taken together, we have provided an extended potency, safety, and molecular profile of FReP cell-based bone regeneration. Therefore, FReP cells present a high potential for cellular and gene therapy products for bone regeneration. PMID:26774565

  8. 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.

  9. Characterization studies of suppressor cells in murine bone marrow chimaeras.

    PubMed Central

    Imamura, M; Fujimoto, H; Fukuhara, T; Kobayashi, M; Kasai, M; Sakurada, K; Miyazaki, T

    1987-01-01

    When BALB/c bone marrow cells were transferred to lethally X-irradiated C3H/He mice either intrasplenically (i.s.) or intravenously (i.v.), suppressor cells were detected by means of MLR coculture assays in the spleen of i.s. and i.v. chimaeras. Some but not all of the suppressor cells expressed a Thy 1.2 antigen, indicating that suppressor cells either sensitive or insensitive to anti-Thy 1.2 antibody plus complement treatment were generated in the spleen of i.s. and i.v. chimaeras. According to the examination of Lyt alloantigen expression on suppressor T cells, Lyt 1+2-, Lyt 1-2+, and Lyt 1+2+ suppressor T cells appeared to be present. The culture supernatants from several T-cell clones showed the suppressor activity against alloreactive MLR. Cell surface markers of these clones were composed of Lyt 1+2-, Lyt 1+2+ and Lyt 1-2+. In addition, since Carrageenan treatment abrogated the suppressor activity of plastic dish adherent cells, we conclude that some of them were composed mainly of macrophages. PMID:2950049

  10. Immunosuppressive properties of cloned bone marrow mesenchymal stem cells.

    PubMed

    Xu, Guangwu; Zhang, Liying; Ren, Guangwen; Yuan, Zengrong; Zhang, Yingyu; Zhao, Robert C; Shi, Yufang

    2007-03-01

    Mesenchymal stem cells (MSCs), derived from adult tissues, are multipotent progenitor cells, which hold great promise for regenerative medicine. Recent studies have shown that MSCs are immunosuppressive in vivo and in vitro in both animals and humans. However, the mechanisms that govern these immune modulatory functions of MSCs remain largely elusive. Some studies with bulk populations of MSCs indicated that soluble factors such as PGE2 and TGFbeta are important, while others support a role for cell-cell contact. In this study, we intended to clarify these issues by examining immunosuppressive effects of cloned MSCs. We derived MSC clones from mouse bone marrow and showed that the majority of these clones were able to differentiate into adipocytes and osteoblast-like cells. Importantly, cells from these clones exhibited strong inhibitory effects on TCR activation-induced T cell proliferation in vitro, and injection of a small number of these cells promoted the survival of allogeneic skin grafts in mice. Conditioned medium from MSC cultures showed some inhibitory effect on anti-CD3 induced lymphocyte proliferation independent of PGE2 and TGFbeta. In comparison, direct co-culture of MSCs with stimulated lymphocytes resulted in much stronger immunosuppressive effect. Interestingly, the suppression was bi-directional, as MSC proliferation was also reduced in the presence of lymphocytes. Taking together, our findings with cloned MSCs demonstrate that these cells exert their immunosuppressive effects through both soluble factor(s) and cell-cell contact, and that lymphocytes and MSCs are mutually inhibitory on their respective proliferation. PMID:17325691

  11. 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

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

    PubMed

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

    2012-06-01

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

  13. 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

  14. Cell seeding density is a critical determinant for copolymer scaffolds-induced bone regeneration.

    PubMed

    Yassin, Mohammed A; Leknes, Knut N; Pedersen, Torbjorn O; Xing, Zhe; Sun, Yang; Lie, Stein A; Finne-Wistrand, Anna; Mustafa, Kamal

    2015-11-01

    Constructs intended for bone tissue engineering (TE) are influenced by the initial cell seeding density. Therefore, the objective of this study was to determine the effect of bone marrow stromal stem cells (BMSCs) density loaded onto copolymer scaffolds on bone regeneration. BMSCs were harvested from rat's bone marrow and cultured in media with or without osteogenic supplements. Cells were seeded onto poly(l-lactide-co-ε-caprolactone) [poly(LLA-co-CL)] scaffolds at two different densities: low density (1 × 10(6) cells/scaffold) or high density (2 × 10(6) cells/scaffold) using spinner modified flasks and examined after 1 and 3 weeks. Initial attachment and spread of BMSC onto the scaffolds was recorded by scanning electron microscopy. Cell proliferation was assessed by DNA quantification and cell differentiation by quantitative real-time reverse transcriptase-polymerized chain reaction analysis (qRT-PCR). Five-millimeter rat calvarial defects (24 defects in 12 rats) were implanted with scaffolds seeded with either low or high density expanded with or without osteogenic supplements. Osteogenic supplements significantly increased cell proliferation (p < 0.001). Scaffolds seeded at high cell density exhibited higher mRNA expressions of Runx2 p = 0.001, Col1 p = 0.001, BMP2 p < 0.001, BSP p < 0.001, and OC p = 0.013. More bone was formed in response to high cell seeding density (p = 0.023) and high seeding density with osteogenic medium (p = 0.038). Poly (LLA-co-CL) scaffolds could be appropriate candidates for bone TE. The optimal number of cells to be loaded onto scaffolds is critical for promoting Extracellular matrix synthesis and bone formation. Cell seeding density and osteogenic supplements may have a synergistic effect on the induction of new bone. PMID:26013960

  15. Communication of bone cells with hematopoiesis, immunity and energy metabolism.

    PubMed

    Asada, Noboru; Sato, Mari; Katayama, Yoshio

    2015-01-01

    The bone contains the bone marrow. The functional communication between bone cells and hematopoiesis has been extensively studied in the past decade or so. Osteolineage cells and their modulators, such as the sympathetic nervous system, macrophages and osteoclasts, form a complex unit to maintain the homeostasis of hematopoiesis, called the 'microenvironment'. Recently, bone-embedded osteocytes, the sensors of gravity and mechanical stress, have joined the microenvironment, and they are demonstrated to contribute to whole body homeostasis through the control of immunity and energy metabolism. The inter-organ communication orchestrated by the bone is summarized in this article. PMID:26512322

  16. The Alliance of Mesenchymal Stem Cells, Bone, and Diabetes

    PubMed Central

    Napoli, Nicola; Paladini, Angela; Briganti, Silvia I.; Pozzilli, Paolo; Epstein, Sol

    2014-01-01

    Bone fragility has emerged as a new complication of diabetes. Several mechanisms in diabetes may influence bone homeostasis by impairing the action between osteoblasts, osteoclasts, and osteocytes and/or changing the structural properties of the bone tissue. Some of these mechanisms can potentially alter the fate of mesenchymal stem cells, the initial precursor of the osteoblast. In this review, we describe the main factors that impair bone health in diabetic patients and their clinical impact. PMID:25140176

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

    PubMed

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

    2011-01-01

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

  18. 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

  19. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication

    SciTech Connect

    Hock, J.M.; Centrella, M.; Canalis, E.

    1988-01-01

    The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of (2,3-/sup 3/H)proline and (methyl-/sup 3/H)thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on (/sup 3/H)proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on (/sup 3/H)thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis.

  20. 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

  1. 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

  2. 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

  3. Intraosseous injection of RM1 murine prostate cancer cells promotes rapid osteolysis and periosteal bone deposition

    PubMed Central

    McCabe, N. Patrick; Madajka, Maria; Vasanji, Amit

    2010-01-01

    The molecular mechanisms associated with prostate cancer (PCa) progression within bone remain a topic of intense investigation. With the availability of transgenic mouse strains, a model of PCa for use in immune competent/transgenic mice would be highly beneficial. This study was designed to explore the utility of RM1 mouse PCa cells in investigations of tumor:bone interactions. The efficacies of several implantation techniques were examined for reliably producing intra-bone RM1 tumor growth and bone lesion formation in immune competent mice. Longitudinal monitoring of bone remodeling and lesion phenotypes was conducted by microcomputed tomography (μCT) and histological analyses. Our results indicate that direct intrabone injections of RM1 cells are necessary for tumor growth within bone and direct implantation promotes the rapid development of osteolytic bone lesions with periosteal bone deposition post-cortical breach. In vitro, RM1 cells promote the proliferation of osteoblast (MC3T3-E1) and osteoclast (Raw264.7) progenitors in a dose dependent manner. Conditioned culture media from RM1 cells appears to promote earlier expression of genes/proteins associated with osteoblastic differentiation. While clearly stimulating osteoclast function in vivo, RM1 cells had little effect on differentiation and tartate resistant acid phosphatase (TRAP) expression by Raw264.7 cells. These data, coupled with in vivo μCT images, indicate the ability of RM1 cells to induce mixed, yet predominentally osteolytic, responses in bone and illustrate the potential of RM1 cells as a model of investigating prostate tumor:stroma interactions in immune competent/transgenic mice on a C57BL/6 background. PMID:18506587

  4. 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

  5. Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma.

    PubMed

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

    2012-06-01

    Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor 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. 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

  6. 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

  7. Effects of prostaglandin E3 and eicosapentaenoic acid on rat bone in organ culture.

    PubMed

    Raisz, L G; Alander, C B; Simmons, H A

    1989-05-01

    To assess the possibility that diets rich in eicosapentaenoic acid (EPA) could have adverse effects on the skeleton, we examined the resorptive response to its major project, PGE3, and the effects and metabolism of EPA itself in cultured fetal rat long bones and neonatal rat calvaria. PGE3 stimulated bone resorption with a potency similar to that of PGE2. However, EPA was a much less effective precursor for PGE3 than was arachidonic acid (AA) for PGE2. In bones cultured with complement sufficient rabbit serum, which stimulates endogenous PGE release, addition of EPA had little effect on bone resorption while AA produced a substantial increase. Bones labeled with [3H]-AA and incubated with transforming growth factor-alpha (TGF-alpha), which stimulates endogenous PGE production, produced substantial amounts of PGE2, while bones labeled with [3H]-EPA and treated similarly produced less than 1/10th as much labeled PGE3. Thus, EPA appears to be a less effective precursor for the production of bone resorbing prostanoids than AA in cultured rat bone. However, since PGE3 is a potent stimulator of bone resorption, the possibility that dietary EPA can effect the production of bone resorbing prostanoids in man requires further study. PMID:2544927

  8. Multipotent adult progenitor cell-loaded demineralized bone matrix for bone tissue engineering.

    PubMed

    Supronowicz, Peter; Gill, Elise; Trujillo, Angelica; Thula, Taili; Zhukauskas, Rasa; Perry, Robert; Cobb, Ronald R

    2016-04-01

    Multipotent adult progenitor cells (MAPCs) from bone marrow have been shown to be capable of forming bone, cartilage and other connective tissues. In addition, MAPCs differentiate into lineages that are different from their germ layers of origin. Previous studies showed the ability of MAPCs to improve cardiac function and control allogenic-reactive responses associated with acute graft versus host disease. In the current study, we evaluated the ability of MAPCs to produce bone matrix on demineralized bone allograft substrates. Specifically, MAPCs expressed alkaline phosphatase, produced extracellular matrix proteins and deposited calcium-containing mineral on demineralized bone matrices. Furthermore, the addition of MAPCs on demineralized bone matrix (DBM) scaffolds enhanced osteoinductivity of the carrier in a rat ectopic pouch model. These results demonstrated the potential of MAPCs as a new approach for bone repair in tissue-engineering applications. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23413005

  9. CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

    SciTech Connect

    Oue, Erika; Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University; Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo ; Lee, Ji-Won; Sakamoto, Kei; Iimura, Tadahiro; Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo ; Aoki, Kazuhiro; Kayamori, Kou; Department of Pathology, Ome Municipal General Hospital, Ome, Tokyo ; Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo; Yamaguchi, Akira; Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo

    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 report showing the role of CXCL2 in cancer-associated bone destruction.

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

    PubMed

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

    2014-07-01

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

  11. Bone marrow–derived progenitor cells in pulmonary fibrosis

    PubMed Central

    Hashimoto, Naozumi; Jin, Hong; Liu, Tianju; Chensue, Stephen W.; Phan, Sem H.

    2004-01-01

    The origin of fibroblasts in pulmonary fibrosis is assumed to be intrapulmonary, but their extrapulmonary origin and especially derivation from bone marrow (BM) progenitor cells has not been ruled out. To examine this possibility directly, adult mice were durably engrafted with BM isolated from transgenic mice expressing enhanced GFP. Induction of pulmonary fibrosis in such chimera mice by endotracheal bleomycin (BLM) injection caused large numbers of GFP+ cells to appear in active fibrotic lesions, while only a few GFP+ cells could be identified in control lungs. Flow-cytometric analysis of lung cells confirmed the BLM-induced increase in GFP+ cells in chimera mice and revealed a significant increase in GFP+ cells that also express type I collagen. GFP+ lung fibroblasts isolated from chimera mice expressed collagen and telomerase reverse transcriptase but not α-smooth muscle actin. Treatment of isolated GFP+ fibroblasts with TGF-β failed to induce myofibroblast differentiation. Cultured lung fibroblasts expressed the chemokine receptors CXCR4 and CCR7 and responded chemotactically to their cognate ligands, stromal cell–derived factor-1α and secondary lymphoid chemokine, respectively. Thus the collagen-producing lung fibroblasts in pulmonary fibrosis can also be derived from BM progenitor cells. PMID:14722616

  12. 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

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

    PubMed Central

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

    2013-01-01

    JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies. PMID:23805194

  14. 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.

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

    PubMed Central

    Khatri, Mahesh; Saif, Yehia M.

    2011-01-01

    The bone marrow contains heterogeneous population of cells that are involved in the regeneration and repair of diseased organs, including the lungs. In this study, we isolated and characterized progenitor epithelial cells from the bone marrow of 4- to 5-week old germ-free pigs. Microscopically, the cultured cells showed epithelial-like morphology. Phenotypically, these cells expressed the stem cell markers octamer-binding transcription factor (Oct4) and stage-specific embryonic antigen-1 (SSEA-1), the alveolar stem cell marker Clara cell secretory protein (Ccsp), and the epithelial cell markers pan-cytokeratin (Pan-K), cytokeratin-18 (K-18), and occludin. When cultured in epithelial cell growth medium, the progenitor epithelial cells expressed type I and type II pneumocyte markers. Next, we examined the susceptibility of these cells to influenza virus. Progenitor epithelial cells expressed sialic acid receptors utilized by avian and mammalian influenza viruses and were targets for influenza virus replication. Additionally, differentiated type II but not type I pneumocytes supported the replication of influenza virus. Our data indicate that we have identified a unique population of progenitor epithelial cells in the bone marrow that might have airway reconstitution potential and may be a useful model for cell-based therapies for infectious and non-infectious lung diseases. PMID:22216319

  16. Production of interleukin-6 by human osteoclast-like cells from giant cell tumor of bone.

    PubMed

    Giovannini, M; Strippoli, P; Serra, M; Sironi, M; Fincato, G; Colotta, F; Bonafe, M; Biunno, I; Mantovani, A; Orlandini, S; Brandi, M; Pastano, R; Bagnara, G

    1996-02-01

    Giant cell tumor (GCT) is a bone neoplasm which is characterized by the presence of large numbers of multinucleated osteoclast-like giant cells. Although GCT can be considered a benign lesion, it exhibits high local aggressiveness often associated with osteolytic properties. In this study, we used five different GCT primary cell cultures to evaluate whether osteoclast-like cells from GCT are able to produce interleukin-6 (IL-6), a cytokine strictly involved in the induction of osteoclast-mediated bone resorption. IL-6 assessment with ELISA revealed that osteoclast-like GCT cells produce low levels of this cytokine, which can be greatly increased after treatment with both lipopolysaccharide (LPS) or interleukin-1 beta (IL-1 beta). These data were confirmed by molecular analysis which revealed that GCT cells synthesize IL-6 mRNA and that the levels of IL-6 transcripts are greatly increased after treatment with both LPS and IL-1 beta. Moreover, by using a biologic assay with the 7TD1, a IL-6 dependent cell Line, we also determined that IL-6 synthesized by GCT cells is biologically active. This study supports the hypothesis that IL-6 locally released by GCT osteoclast-like cells may be involved in the induction of the osteolysis which is strictly associated with the biologic aggressiveness of GCT cells. PMID:21544359

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

    PubMed

    Chen, Tao; Wang, Yanhui; Bu, Lingxue; Li, Ningyi

    2014-04-01

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

  18. Cell-Based Approaches to the Engineering of Vascularized Bone Tissue

    PubMed Central

    Rao, Rameshwar R.; Stegemann, Jan P.

    2013-01-01

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

  19. Preclinical models for in vitro mechanical loading of bone-derived cells.

    PubMed

    Michael Delaine-Smith, Robin; Javaheri, Behzad; Helen Edwards, Jennifer; Vazquez, Marisol; Rumney, Robin Mark Howard

    2015-01-01

    It is well established that bone responds to mechanical stimuli whereby physical forces are translated into chemical signals between cells, via mechanotransduction. It is difficult however to study the precise cellular and molecular responses using in vivo systems. In vitro loading models, which aim to replicate forces found within the bone microenvironment, make the underlying processes of mechanotransduction accessible to the researcher. Direct measurements in vivo and predictive modeling have been used to define these forces in normal physiological and pathological states. The types of mechanical stimuli present in the bone include vibration, fluid shear, substrate deformation and compressive loading, which can all be applied in vitro to monolayer and three-dimensional (3D) cultures. In monolayer, vibration can be readily applied to cultures via a low-magnitude, high-frequency loading rig. Fluid shear can be applied to cultures in multiwell plates via a simple rocking platform to engender gravitational fluid movement or via a pump to cells attached to a slide within a parallel-plate flow chamber, which may be micropatterned for use with osteocytes. Substrate strain can be applied via the vacuum-driven FlexCell system or via a four-point loading jig. 3D cultures better replicate the bone microenvironment and can also be subjected to the same forms of mechanical stimuli as monolayer, including vibration, fluid shear via perfusion flow, strain or compression. 3D cocultures that more closely replicate the bone microenvironment can be used to study the collective response of several cell types to loading. This technical review summarizes the methods for applying mechanical stimuli to bone cells in vitro. PMID:26331007

  20. Cell kinetics and longitudinal bone growth in birds.

    PubMed

    Kember, N F; Kirkwood, J K

    1987-11-01

    The theory that links cell division in epiphyseal cartilage plates to overall growth of long bones has been extended from linear growth systems to those in which proliferating and hypertrophied cells are not arranged in columns. Consideration has also been given to the analysis of non-parallel growth systems. The theory is illustrated by examples from the growth of chicken bones. PMID:3502931

  1. Integrins and bone metastasis: Integrating tumor cell and stromal cell interactions

    PubMed Central

    Schneider, Jochen G.; Amend, Sarah H.; Weilbaecher, Katherine N.

    2010-01-01

    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 ?v?3 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 ?v?3 integrins are critical to osteoclast 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 play critical roles in platelet aggregation (?v?3 and ?IIb?3), hematopoietic cell mobilization (VLA-4, osteopontin), neoangiogenesis (?v?3,?v?5, ?6?4, ?1 integrin) and stromal function (osteopontin, 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. PMID:20850578

  2. Developmental cues for bone formation from parathyroid hormone and parathyroid hormone-related protein in an ex vivo organotypic culture system of embryonic chick femora.

    PubMed

    Smith, Emma L; Kanczler, Janos M; Roberts, Carol A; Oreffo, Richard O C

    2012-12-01

    Enhancement and application of our understanding of skeletal developmental biology is critical to developing tissue engineering approaches to bone repair. We propose that use of the developing embryonic femur as a model to further understand skeletogenesis, and the effects of key differentiation agents, will aid our understanding of the developing bone niche and inform bone reparation. We have used a three-dimensional organotypic culture system of embryonic chick femora to investigate the effects of two key skeletal differentiation agents, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), on bone and cartilage development, using a combination of microcomputed tomography and histological analysis to assess tissue formation and structure, and cellular behavior. Stimulation of embryonic day 11 (E11) organotypic femur cultures with PTH and PTHrP initiated osteogenesis. Bone formation was enhanced, with increased collagen I and STRO-1 expression, and cartilage was reduced, with decreased chondrocyte proliferation, collagen II expression, and glycosaminoglycan levels. This study demonstrates the successful use of organotypic chick femur cultures as a model for bone development, evidenced by the ability of exogenous bioactive molecules to differentially modulate bone and cartilage formation. The organotypic model outlined provides a tool for analyzing key temporal stages of bone and cartilage development, providing a paradigm for translation of bone development to improve scaffolds and skeletal stem cell treatments for skeletal regenerative medicine. PMID:22690868

  3. [The application progress of human urine derived stem cells in bone tissue engineering].

    PubMed

    Gao, P; Jiang, D P; Li, Z Z

    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

  4. Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in diseases of the skeleton

    PubMed Central

    Chanda, Diptiman; Kumar, Sanjay; Ponnazhagan, Selvarangan

    2010-01-01

    Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late sixties and early seventies, MSCs or MSC-like cells have been obtained from various mesodermal and non-mesodermal tissues, although majority of the therapeutic applications involved bone marrow derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis respectively and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long-term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone-related diseases. PMID:20506559

  5. [Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat].

    PubMed

    Byeverova, E I; Bragina, E V; Molchanova, E A

    2008-01-01

    The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells. PMID:19137707

  6. In vivo bone formation by progeny of human embryonic stem cells.

    PubMed

    Kuznetsov, Sergei A; Cherman, Natasha; Robey, Pamela Gehron

    2011-02-01

    The derivation of osteogenic cells from human embryonic stem cells (hESCs) or from induced pluripotent stem cells for bone regeneration would be a welcome alternative to the use of adult stem cells. In an attempt to promote hESC osteogenic differentiation, cells of the HSF-6 line were cultured in differentiating conditions in vitro for prolonged periods of time ranging from 7 to 14.5 weeks, followed by in vivo transplantation into immunocompromised mice in conjunction with hydroxyapatite/tricalcium phosphate ceramic powder. Twelve different medium compositions were tested, along with a number of other variables in culture parameters. In differentiating conditions, HSF-6-derived cells demonstrated an array of diverse phenotypes reminiscent of multiple tissues, but after a few passages, acquired a more uniform, fibroblast-like morphology. Eight to 16 weeks post-transplantation, a group of transplants revealed the formation of histologically proven bone of human origin, including broad areas of multiple intertwining trabeculae, which represents by far the most extensive in vivo bone formation by the hESC-derived cells described to date. Knockout-Dulbecco's modified Eagle's medium-based media with fetal bovine serum, dexamethasone, and ascorbate promoted more frequent bone formation, while media based on ?-modified minimum essential medium promoted teratoma formation in 12- to 20-week-old transplants. Transcription levels of pluripotency-related (octamer binding protein 4, Nanog), osteogenesis-related (collagen type I, Runx2, alkaline phosphatase, and bone sialoprotein), and chondrogenesis-related (collagen types II and X, and aggrecan) genes were not predictive of either bone or teratoma formation. The most extensive bone was formed by the strains that, following 4 passages in monolayer conditions, were cultured for 23 to 25 extra days on the surface of hydroxyapatite/tricalcium phosphate particles, suggesting that coculturing of hESC-derived cells with osteoconductive material may increase their osteogenic potential. While none of the conditions tested in this study, and elsewhere, ensured consistent bone formation by hESC-derived cells, our results may elucidate further directions toward the construction of bone on the basis of hESCs or an individual's own induced pluripotent stem cells. PMID:20590404

  7. 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

  8. Osteogenic differentiation of human bone marrow mesenchymal stem cells seeded on melt based chitosan scaffolds for bone tissue engineering applications.

    PubMed

    Costa-Pinto, Ana R; Correlo, Vitor M; Sol, Paula C; Bhattacharya, Mrinal; Charbord, Pierre; Delorme, Bruno; Reis, Rui L; Neves, Nuno M

    2009-08-10

    The purpose of this study was to evaluate the growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded onto new biodegradable chitosan/polyester scaffolds. Scaffolds were obtained by melt blending chitosan with poly(butylene succinate) in a proportion of 50% (wt) each and further used to produce a fiber mesh scaffold. hBMSCs were seeded on those structures and cultured for 3 weeks under osteogenic conditions. Cells were able to reduce MTS and demonstrated increasing metabolic rates over time. SEM observations showed cell colonization at the surface as well as within the scaffolds. The presence of mineralized extracellular matrix (ECM) was successfully demonstrated by peaks corresponding to calcium and phosphorus elements detected in the EDS analysis. A further confirmation was obtained when carbonate and phosphate group peaks were identified in Fourier Transformed Infrared (FTIR) spectra. Moreover, by reverse transcriptase (RT)-PCR analysis, it was observed the expression of osteogenic gene markers, namely, Runt related transcription factor 2 (Runx2), type 1 collagen, bone sialoprotein (BSP), and osteocalcin. Chitosan-PBS (Ch-PBS) biodegradable scaffolds support the proliferation and osteogenic differentiation of hBMSCs cultured at their surface in vitro, enabling future in vivo testing for the development of bone tissue engineering therapies. PMID:19621927

  9. 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

  10. Mesenchymal stem cells: mechanisms and role in bone regeneration.

    PubMed

    Qin, Yunhao; Guan, Junjie; Zhang, Changqing

    2014-11-01

    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

  11. 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 ligand-bone marrow-derived dendritic cells mostly resemble classic dendritic cells but comprise additional minor subpopulations, whereas GM-CSF/IL-4-bone marrow-derived dendritic cells resemble monocyte-derived inflammatory dendritic cells (iNOS-positive monocyte-derived cells). PMID:26516183

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

    PubMed Central

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

    2011-01-01

    Circulating bone marrow-derived immature cells, including endothelial progenitor cells, have been implicated in homeostasis of the microvasculature. Decreased levels of circulating endothelial progenitor cells, associated with aging and/or cardiovascular risk factors, correlate with poor clinical outcomes in a range of cardiovascular diseases. Herein, we transplanted bone marrow cells from young stroke-prone spontaneously hypertensive rats (SHR-SP) into aged SHR-SP, the latter not exposed to radiation or chemotherapy. Analysis of recipient peripheral blood 28 days after transplantation revealed that 5% of circulating blood cells were of donor origin. Cerebral infarction was induced on day 30 posttransplantation. Animals transplanted with bone marrow from young SHR-SP displayed an increase in density of the microvasculature in the periinfarction zone, reduced ischemic brain damage and improved neurologic function. In vitro analysis revealed enhanced activation of endothelial nitric oxide synthase and reduced activation p38 microtubule-associated protein (MAP) kinase, the latter associated with endothelial apoptosis, in cultures exposed to bone marrow-derived mononuclear cells from young animals versus cells from aged counterparts. Our findings indicate that partial rejuvenation of bone marrow from aged rats with cells from young animals enhances the response to ischemic injury, potentially at the level of endothelial/vascular activation, providing insight into a novel approach ameliorate chronic vascular diseases. PMID:20859292

  13. Bioreactor Strategy in Bone Tissue Engineering: Pre-Culture and Osteogenic Differentiation Under Two Flow Configurations

    PubMed Central

    Kim, Junho

    2012-01-01

    Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both pre-culture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome. PMID:22690750

  14. 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 cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K. PMID:26426806

  15. Artificial Niche Combining Elastomeric Substrate and Platelets Guides Vascular Differentiation of Bone Marrow Mononuclear Cells

    PubMed Central

    Wu, Wei; Allen, Robert; Gao, Jin

    2011-01-01

    Bone marrow-derived progenitor cells are promising cell sources for vascular tissue engineering. However, conventional bone marrow mesenchymal stem cell expansion and induction strategies require plating on tissue culture plastic, a stiff substrate that may itself influence cell differentiation. Direct scaffold seeding avoids plating on plastic; to the best of our knowledge, there is no report of any scaffold that induces the differentiation of bone marrow mononuclear cells (BMNCs) to vascular cells in vitro. In this study, we hypothesize that an elastomeric scaffold with adsorbed plasma proteins and platelets will induce differentiation of BMNCs to vascular cells and promote vascular tissue formation by combining soft tissue mechanical properties with platelet-mediated tissue repairing signals. To test our hypothesis, we directly seeded rat primary BMNCs in four types of scaffolds: poly(lactide-co-glycolide), elastomeric poly(glycerol sebacate) (PGS), platelet-poor plasma-coated PGS, and PGS coated by plasma supplemented with platelets. After 21 days of culture, osteochondral differentiation of cells in poly(lactide-co-glycolide) was detected, but most of the adhered cells on the surface of all PGS scaffolds expressed calponin-I and α-smooth muscle actin, suggesting smooth muscle differentiation. Cells in PGS scaffolds also produced significant amount of collagen and elastin. Further, plasma coating improves seeding efficiency, and platelet increases proliferation, the number of differentiated cells, and extracellular matrix content. Thus, the artificial niche composed of platelets, plasma, and PGS is promising for artery tissue engineering using BMNCs. PMID:21449713

  16. Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models.

    PubMed

    Liu, Jess; Czernick, Drew; Lin, Shih-Chun; Alasmari, Abeer; Serge, Dibart; Salih, Erdjan

    2013-09-01

    Egg yolk phosvitin is one of the most highly phosphorylated extracellular matrix proteins known in nature with unique physico-chemical properties deemed to be critical during ex-vivo egg embryo development. We have utilized our unique live mouse calvarial bone organ culture models under conditions which dissociates the two bone remodeling stages, viz., resorption by osteoclasts and formation by osteoblasts, to highlight important and to date unknown critical biological functions of egg phosvitin. In our resorption model live bone cultures were grown in the absence of ascorbate and were stimulated by parathyroid hormone (PTH) to undergo rapid osteoclast formation/differentiation with bone resorption. In this resorption model native phosvitin potently inhibited PTH-induced osteoclastic bone resorption with simultaneous new osteoid/bone formation in the absence of ascorbate (vitamin C). These surprising and critical observations were extended using the bone formation model in the absence of ascorbate and in the presence of phosvitin which supported the above results. The results were corroborated by analyses for calcium release or uptake, tartrate-resistant acid phosphatase activity (marker for osteoclasts), alkaline phosphatase activity (marker for osteoblasts), collagen and hydroxyproline composition, and histological and quantitative histomorphometric evaluations. The data revealed that the discovered bioactivity of phosvitin mirrors that of ascorbate during collagen synthesis and the formation of new osteoid/bone. Complementing those studies use of the synthetic collagen peptide analog and cultured calvarial osteoblasts in conjunction with mass spectrometric analysis provided results that augmented the bone organ culture work and confirmed the capacity of phosvitin to stimulate differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization. There are striking implications and interrelationships of this affect that relates to the evolutionary inactivation of the gene of an enzyme L-gulono-γ-lactone oxidase, which is involved in the final step of ascorbate biosynthesis, in many vertebrate species including passeriform birds, reptiles and teleost fish whose egg yolk contain phosvitin. These represent examples of how developing ex-vivo embryos of such species can achieve connective tissue and skeletal system formation in the absence of ascorbate. PMID:23791550

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

    PubMed

    Siggelkow, H; Hilmes, D; Robenstorff, K; Kurre, W; Engel, I; Hüfner, M

    1998-04-27

    We describe the development of flowcytometrical methods to analyse human primary osteoblast-like cultures obtained from trabecular bone explants in comparison to the human osteosarcoma cell line HOS 58. Two antigens typical of osteoblasts were studied: bone alkaline phosphatase and collagen/procollagen I; the non-specific attachment protein fibronectin served as control. The morphology of all different antigens is shown by immunocytochemistry before flowcytometrical analysis. The establishment of flowcytometry is described in detail. While all antigens tested were nearly 100% positive in the HOS 58 cells in immunocytochemistry and flowcytometry, in primary osteoblast-like cells results varied widely between both methods. Cell permeabilisation before flowcytometry improved the homogeneity of results, probably by increasing the accessibility of the specific antibody to intracellular compartments. Though up to 80% of cells were lost during preparation the ratio of positive versus negative cells in specific experiment was not dependent on the cell recovery. Therefore, the cells finally analysed seemed to be representative of the total population. PMID:9641353

  18. EpCAM expression in breast cancer cells is associated with enhanced bone metastasis formation.

    PubMed

    Hiraga, Toru; Ito, Susumu; Nakamura, Hiroaki

    2016-04-01

    Epithelial cell adhesion molecule (EpCAM) has been implicated in multiple cellular functions including cell adhesion. EpCAM has also recently been identified as a marker for cancer stem cells (CSCs). Here, we examined the roles of EpCAM in the development of bone metastasis of breast cancer by using well-characterized animal models. Morphological and real-time reverse transcriptase-polymerase chain reaction data showed that the EpCAM-negative and -positive (EpCAM(neg) and EpCAM(pos) ) cell populations isolated from breast cancer cell lines exhibited mesenchymal and epithelial phenotypes, respectively. Flow cytometric analysis revealed that EpCAM(pos) , but not EpCAM(neg) , cells possessed self-renewal and differentiation potentials. Tumorsphere formation in suspension cultures and tumorigenicity in the orthotopic mammary fat pad of mice were significantly greater in EpCAM(pos) cells than in EpCAM(neg) cells. The development of bone metastases induced by an intracardiac injection was markedly increased in mice inoculated with EpCAM(pos) cells. Furthermore, intracardiac inoculations of parental cells demonstrated that the EpCAM(pos) population in cancer cells that colonized in bone was significantly higher than that in parental cells. However, stable transduction of EpCAM into EpCAM(neg) cells failed to reproduce the phenotypes of EpCAM(pos) cells. These results suggest that the expression of EpCAM in breast cancer cells is associated with CSC-like phenotypes, which contribute to the promotion of bone metastases by enhancing tumorigenicity. Our results also support the possibility that the epithelial phenotypes of EpCAM-expressing cells confer advantageous properties for the development of bone metastases, at least after entering the circulation, while EpCAM is likely not solely responsible for the phenotypes of EpCAM(pos) cells. PMID:26576938

  19. Development of silk-based scaffolds for tissue engineering of bone from human adipose derived stem cells

    PubMed Central

    Correia, Cristina; Bhumiratana, Sarindr; Yan, Le-Ping; Oliveira, Ana L.; Gimble, Jeffrey M.; Rockwood, Danielle; Kaplan, David L.; Sousa, Rui A.; Reis, Rui L.; Vunjak-Novakovic, Gordana

    2012-01-01

    Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and demonstrated ability to support bone formation, in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASC), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol - HFIP), pore sizes (250–500μm vs. 500–1000μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes with decellularized trabecular bone (DCB) included as a “gold standard”. The scaffolds were seeded with hASC and cultured for 7 weeks in osteogenic media. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2, and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation. PMID:22421311

  20. A thixotropic nanocomposite gel for three-dimensional cell culture

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

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

  1. Applicability of human dental follicle cells to bone regeneration without dexamethasone: an in vivo pilot study.

    PubMed

    Takahashi, K; Ogura, N; Tomoki, R; Eda, T; Okada, H; Kato, R; Iwai, S; Ito, K; Kuyama, K; Kondoh, T

    2015-05-01

    The aim of this study was to investigate the capacity of human dental follicle cells (hDFCs) for bone formation in vivo. hDFCs were obtained from wisdom teeth extracted from patients aged 14 and 22 years. hDFCs from the 5th to 8th passages were grown in three-dimensional (3D) culture using gelatin sponges. Cells were transplanted onto the calvaria of F344/NJcl-rnu/rnu male rats (immunodeficient rats). Haematoxylin and eosin (HE) staining and immunohistochemistry were performed, and newly formed bone was evaluated by micro-computed tomography (micro-CT). HE staining showed newly formed bone in 3D culture. Immunohistochemistry showed bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX2), and osterix staining in areas with newly formed bone. Furthermore, micro-CT showed that, in comparison to controls, transplanted hDFCs promoted better bone quality and bone mineral density (BMD 582 ± 131.1 vs. 300.5 ± 77.7 mg/cm(3); P=0.039), bone mineral content (BMC 5.6 ± 1.1 vs. 2.1 ± 0.4 mg; P = 0.006), bone volume (BV 9.7 ± 0.5 × 10(-3) vs. 7.0 ± 0.4 × 10(-3) cm(3); P = 0.002), BMC/total volume (TV) (399.9 ± 76.3 vs. 147.7 ± 30.8 mg/cm(3); P = 0.006), and BV/TV (69.1 ± 3.6% vs. 49.6 ± 3.1%; P=0.002). This suggests that human dental follicles are potentially useful for regenerative therapy. PMID:25496849

  2. Bone pulsating metastasis due to renal cell carcinoma.

    PubMed

    Cınar, Murat; Derincek, Alihan; Karan, Belgin; Akpınar, Sercan; Tuncay, Cengiz

    2010-11-01

    Pulsation on the bone cortex surface is a rare condition. Pulsative palpation of the superficial-located bone tumors can be misperceived as an aneurysm. Fifty-eight-year-old man is presented with pulsating bone mass in his proximal tibia. During angiographic examination, hypervascular masses were diagnosed both at right kidney and at right proximal tibia. Renal cell carcinoma was diagnosed after abdominal CT scan. Proximal tibia biopsy was complicated with projectile bleeding. PMID:20376589

  3. Mechanical characterization of adult stem cells from bone marrow and perivascular niches

    PubMed Central

    Ribeiro, Alexandre J.S.; Tottey, Steven; Taylor, Richard W. E.; Bise, Ryoma; Kanade, Takeo; Badylak, Stephen F.; Dahl, Kris Noel

    2012-01-01

    Therapies using adult stem cells often require mechanical manipulation such as injection or incorporation into scaffolds. However, force-induced rupture and mechanosensitivity of cells during manipulation is largely ignored. Here, we image cell mechanical structures and perform a biophysical characterization of three different types of human adult stem cells: bone marrow CD34+ hematopoietic, bone marrow mesenchymal and perivascular mesenchymal stem cells. We use micropipette aspiration to characterize cell mechanics and quantify deformation of subcellular structures under force and its contribution to global cell deformation. Our results suggest that CD34+ cells are mechanically suitable for injection systems since cells transition from solid- to fluid-like at constant aspiration pressure, probably due to a poorly developed actin cytoskeleton. Conversely, mesenchymal stem cells from the bone marrow and perivascular niches are more suitable for seeding into biomaterial scaffolds since they are mechanically robust and have developed cytoskeletal structures that may allow cellular stable attachment and motility through solid porous environments. Among these, perivascular stem cells cultured in 6% oxygen show a developed cytoskeleton but a more compliant nucleus, which can facilitate the penetration into pores of tissues or scaffolds. We confirm the relevance of our measurements using cell motility and migration assays and measure survival of injected cells. Since different types of adult stem cells can be used for similar applications, we suggest considering mechanical properties of stem cells to match optimal mechanical characteristics of therapies. PMID:22349118

  4. Activation of nervous system development genes in bone marrow derived mesenchymal stem cells following spaceflight exposure.

    PubMed

    Monticone, Massimiliano; Liu, Yi; Pujic, Natalija; Cancedda, Ranieri

    2010-10-01

    Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity-induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA-8 + Increment 13) from March 30 to April 8, 2006 (experiment "Stroma-2"). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo-inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell-cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC. PMID:20658479

  5. Hematopoietic Stem Cell and Its Bone Marrow Niche.

    PubMed

    Yu, V W C; Scadden, D T

    2016-01-01

    Stem cells do not thrive without their niche. The bone marrow microenvironment is where hematopoietic stem cells maintain their cell state while receiving physiological input to modify their activity in response to changing physiological demands. The complexity of the bone marrow microenvironment is being unraveled and indicates that multiple different cell types contribute to the regulation of stem and progenitor cells. Further, it is becoming evident that the bone marrow represents a composite of niches with different components and different functional roles in hematopoiesis. It is now evident that alterations in specific stromal cells that comprise the bone marrow microenvironment can contribute to hematologic pathology. In this chapter, we will review the history of the niche concept, evolving information about its components and how niche dysfunction may contribute to disease. PMID:27137653

  6. Cell Culture as an Alternative in Education.

    ERIC Educational Resources Information Center

    Nardone, Roland M.

    1990-01-01

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

  7. Cell culture techniques in honey bee research

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Photosensitization of cultured cells and viruses by pyrene lipids.

    PubMed

    Gatt, S; Dinur, T; Abou-Rabia, S; Kotler, M; Fibach, E

    1990-12-01

    Administration of pyrene-linked fatty acids and lipids to cultured cells or an enveloped (vesicular stomatitis) virus induced photosensitization which, following irradiation with a long ultra-violet light (LUV), resulted in killing of the cells and loss of the infectivity of the virus with the following specific effects. (i) LUV illumination of the pyrene-sphingomyelin administered cultured skin fibroblasts derived from normal individuals and patients with Niemann-Pick disease permitted selective killing of the latter. (ii) Similarly LUV illumination of pyrenedodecanoic acid (P12) incubates of leukemic cell lines mixed with human bone marrow cells permitted selective killing of the former. (iii) LUV illumination of P12 incubates of vesicular stomatitis virus decreased the infectivity of the virus by up to 12 logs. PMID:1966337

  9. 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

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

    PubMed

    Fu, Yin-Chih; Lin, Chih-Chun; 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

  11. ?-Cell regeneration mediated by human bone marrow mesenchymal stem cells.

    PubMed

    Milanesi, Anna; Lee, Jang-Won; Li, Zhenhua; Da Sacco, Stefano; Villani, Valentina; Cervantes, Vanessa; Perin, Laura; Yu, John S

    2012-01-01

    Bone marrow mesenchymal stem cells (BMSCs) have been shown to ameliorate diabetes in animal models. The mechanism, however, remains largely unknown. An unanswered question is whether BMSCs are able to differentiate into ?-cells in vivo, or whether BMSCs are able to mediate recovery and/or regeneration of endogenous ?-cells. Here we examined these questions by testing the ability of hBMSCs genetically modified to transiently express vascular endothelial growth factor (VEGF) or pancreatic-duodenal homeobox 1 (PDX1) to reverse diabetes and whether these cells were differentiated into ?-cells or mediated recovery through alternative mechanisms. Human BMSCs expressing VEGF and PDX1 reversed hyperglycemia in more than half of the diabetic mice and induced overall improved survival and weight maintenance in all mice. Recovery was sustained only in the mice treated with hBMSCs-VEGF. However, de novo ?-cell differentiation from human cells was observed in mice in both cases, treated with either hBMSCs-VEGF or hBMSCs- PDX1, confirmed by detectable level of serum human insulin. Sustained reversion of diabetes mediated by hBMSCs-VEGF was secondary to endogenous ?-cell regeneration and correlated with activation of the insulin/IGF receptor signaling pathway involved in maintaining ?-cell mass and function. Our study demonstrated the possible benefit of hBMSCs for the treatment of insulin-dependent diabetes and gives new insight into the mechanism of ?-cell recovery after injury mediated by hBMSC therapy. PMID:22879915

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

    SciTech Connect

    Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T.; Jhaveri, Hiral M.; Mishra, Gyan C.; Wani, Mohan R.

    2010-03-12

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

  13. Presentation of a novel model of chitosan- polyethylene oxide-nanohydroxyapatite nanofibers together with bone marrow stromal cells to repair and improve minor bone defects

    PubMed Central

    Emamgholi, Asgar; Rahimi, Mohsen; Kaka, Gholamreza; Sadraie, Seyed Homayoon; Najafi, Saleh

    2015-01-01

    Objective(s): Various methods for repairing bone defects are presented. Cell therapy is one of these methods. Bone marrow stromal cells (BMSCs) seem to be suitable for this purpose. On the other hand, lots of biomaterials are used to improve and repair the defect in the body, so in this study we tried to produce a similar structure to the bone by the chitosan and hydroxyapatite. Materials and Methods: In this study, the solution of chitosan-nanohydroxyapatite-polyethylene oxide (PEO) Nanofibers was produced by electrospinning method, and then the BMSCs were cultured on this solution. A piece of chitosan-nanohydroxyapatite Nanofibers with BMSCs was placed in a hole with the diameter of 1 mm at the distal epiphysis of the rat femur. Then the biomechanical and radiographic studies were performed. Results: Biomechanical testing results showed that bone strength was significantly higher in the Nanofiber/BMSCs group in comparison with control group. Also the bone strength in nanofiber/BMSCs group was significant, but in nanofiber group was nearly significant. Radiographic studies also showed that the average amount of callus formation (radio opacity) in nanofiber and control group was not significantly different. The callus formation in nanofiber/BMSCs group was increased compared to the control group, and it was not significant in the nanofiber group. Conclusion: Since chitosan-nanohydroxyapatite nanofibers with BMSCs increases the rate of bone repair, the obtained cell-nanoscaffold shell can be used in tissue engineering and cell therapy, especially for bone defects. PMID:26523221

  14. Clinical Application of Human Mesenchymal Stromal Cells for Bone Tissue Engineering

    PubMed Central

    Chatterjea, Anindita; Meijer, Gert; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    The gold standard in the repair of bony defects is autologous bone grafting, even though it has drawbacks in terms of availability and morbidity at the harvesting site. Bone-tissue engineering, in which osteogenic cells and scaffolds are combined, is considered as a potential bone graft substitute strategy. Proof-of-principle for bone tissue engineering using mesenchymal stromal cells (MSCs) has been demonstrated in various animal models. In addition, 7 human clinical studies have so far been conducted. Because the experimental design and evaluation parameters of the studies are rather heterogeneous, it is difficult to draw conclusive evidence on the performance of one approach over the other. However, it seems that bone apposition by the grafted MSCs in these studies is observed but not sufficient to bridge large bone defects. In this paper, we discuss the published human clinical studies performed so far for bone-tissue regeneration, using culture-expanded, nongenetically modified MSCs from various sources and extract from it points of consideration for future clinical studies. PMID:21113294

  15. Alterations in bone forming cells due to reduced weight bearing

    NASA Technical Reports Server (NTRS)

    Doty, S. B.; Morey-Holton, E.

    1984-01-01

    A reduction in new bone formation occurred as a result of space flight (Cosmos 1129) and in the suspended animal model of Morey-Holton (1979, 1980). The results indicate that alkaline phosphatase activity of the bone-forming cells is also reduced under these conditions, and the cells in the diaphysis are more affected than those in the metaphyseal region. In addition, these cells show (1) reduced proline incorporation into bone matrix, and (2) increased intracellular lysosomal activity. A change in the cytoskeleton could be the common factor in explaining these results. This suggestion is futher supported by the previous observations that colchicine injections result in decreased osteoblastic function.

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

    NASA Astrophysics Data System (ADS)

    Qin, Sisi; Liao, Wenbin; Ma, Yupo; Simon, Marcia; Rafailovich, Miriam; Stony Brook Medical Center Collaboration; Stony Brook Dental Schoo Collaboration

    2013-03-01

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

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

    PubMed Central

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

    2013-01-01

    Hypothalamic amenorrhea and energy restriction during puberty affect peak bone mass accrual. One hypothesis suggests energy restriction alters hypothalamic function resulting in suppressed estradiol levels leading to bone loss. However, both positive and negative results have been reported regarding energy restriction and bone strength. Therefore, the purpose of this study was to investigate energy restriction and hypothalamic suppression during pubertal onset on bone mechanical strength and the osteogenic capacity of bone marrow-derived cells in two models: female rats treated with gonadotropin releasing hormone antagonists (GnRH-a) or 30% energy restriction. At 23 days of age, female Sprague Dawley rats were assigned to three groups: control group (C, n=10), GnRH-a group (n=10), and Energy Restriction (ER, n=12) group. GnRH-a animals received daily injections for 27 days. The animals in the ER group received 70% of the control animals’ intake. After sacrifice (50 days of age), body weight, uterine and muscle weights were measured. Bone marrow-derived stromal cells were cultured and assayed for proliferation and differentiation into osteoblasts. Outcome measures included bone strength, bone histomorphometry and architecture, serum IGF-1 and osteocalcin. GnRH-a suppressed uterine weight, decreased osteoblast proliferation, bone strength, trabecular bone volume and architecture compared to control. Elevated serum IGF-1 and osteocalcin levels and body weight were found. The ER model had an increase in osteoblast proliferation compared to the GnRH-a group, similar bone strength relative to body weight and increased trabecular bone volume in the lumbar spine compared to control. The ER animals were smaller but had developed bone strength sufficient for their size. In contrast, suppressed estradiol via hypothalamic suppression resulted in bone strength deficits and trabecular bone volume loss. In summary, our results support the hypothesis that during periods of nutritional stress the increased vertebral bone volume may be an adaptive mechanism to store mineral which differs from suppressed estradiol resulting from hypothalamic suppression. PMID:21807131

  18. Multiple melanocortin receptors are expressed in bone cells

    NASA Technical Reports Server (NTRS)

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

    2005-01-01

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

  19. Cell-secreted matrices perpetuate the bone-forming phenotype of differentiated mesenchymal stem cells.

    PubMed

    Hoch, Allison I; Mittal, Vaishali; Mitra, Debika; Vollmer, Nina; Zikry, Christopher A; Leach, J Kent

    2016-01-01

    Prior to transplantation, mesenchymal stem/stromal cells (MSCs) can be induced toward the osteoblastic phenotype using a cocktail of soluble supplements. However, there is little evidence of differentiated MSCs directly participating in bone formation, suggesting that MSCs may either die or revert in phenotype upon transplantation. Cell-secreted decellularized extracellular matrices (DMs) are a promising platform to confer bioactivity and direct cell fate through the presentation of a complex and physiologically relevant milieu. Therefore, we examined the capacity of biomimetic DMs to preserve the mineral-producing phenotype upon withdrawal of the induction stimulus. Regardless of induction duration, ranging up to 6 weeks, MSCs exhibited up to a 5-fold reduction in osteogenic markers within 24 h following stimulus withdrawal. We show that seeding osteogenically induced MSCs on DMs yields up to 2-fold more calcium deposition than tissue culture plastic, and this improvement is at least partially mediated by increasing actin cytoskeletal tension via the ROCK II pathway. MSCs on DMs also secreted 25% more vascular endothelial growth factor (VEGF), a crucial endogenous proangiogenic factor that is abrogated during MSC osteogenic differentiation. The deployment of DMs into a subcutaneous ectopic site enhanced the persistence of MSCs 5-fold, vessel density 3-fold, and bone formation 2-fold more than MSCs delivered without DMs. These results underscore the need for deploying MSCs using biomaterial platforms such as DMs to preserve the in vitro-acquired mineral-producing phenotype and accelerate the process of bone repair. PMID:26457835

  20. Cell Mechanisms of Bone Tissue Loss Under Space Flight Conditions

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia

    Investigations on the space biosatellites has shown that the bone skeleton is one of the most im-portant targets of the effect space flight factors on the organism. Bone tissue cells were studied by electron microscopy in biosamples of rats' long bones flown on the board american station "SLS-2" and in experiments with modelling of microgravity ("tail suspension" method) with using autoradiography. The analysis of data permits to suppose that the processes of remod-eling in bone tissue at microgravity include the following succession of cell-to-cell interactions. Osteocytes as mechanosensory cells are first who respond to a changing "mechanical field". The next stage is intensification of osteolytic processes in osteocytes, leading to a volume en-largement of the osteocytic lacunae and removal of the "excess bone". Then mechanical signals have been transmitted through a system of canals and processes of the osteocytic syncitium to certain superficial bone zones and are perceived by osteoblasts and bone-lining cells (superficial osteocytes), as well as by the bone-marrow stromal cells. The sensitivity of stromal cells, pre-osteoblasts and osteoblasts, under microgravity was shown in a number of works. As a response to microgravity, the system of stromal cells -preosteoblasts -osteoblasts displays retardation of proliferation, differentiation and specific functions of osteogenetic cells. This is supported by the 3H-thymidine studies of the dynamics of differentiation of osteogenetic cells in remodeling zones. But unloading is not adequate and in part of the osteocytes are apoptotic changes as shown by our electron microscopic investigations. An osteocytic apoptosis can play the role in attraction the osteoclasts and in regulation of bone remodeling. The apoptotic bodies with a liquid flow through a system of canals are transferred to the bone surface, where they fulfil the role of haemoattractants for monocytes come here and form osteoclasts. The osteoclasts destroy bone tissue. The macrophages are incorporated into resorption lacunaes and utilize the organic matrix and cellular detritus. The products are secreted to remodeling zones and act as haemoattractants for recruiting and subsequent differentiation here of the osteogenic precursor cells. However, as shown by our results with 3H-glycine, in absence of mechanical stimulus the activization of osteoblastogenesis either doesn't occur, or takes place on a smaller scale. According to our electron-microscopic data a load deficit leads to an adaptive differentiation of fibroblasts and adipocytes in this remodeling zones. This sequence of events is considered as a mechanism of bone tissue loss which underlies the development of osteopenia and osteoporosis under space flight condition.

  1. Scaffolds derived from cancellous bovine bone support mesenchymal stem cells' maintenance and growth.

    PubMed

    Shahabipour, Fahimeh; Mahdavi-Shahri, Nasser; Matin, Maryam M; Tavassoli, Amin; Zebarjad, S Mojtaba

    2013-06-01

    Since bone defects can lead to various disabilities, in recent years, many increasing attempts have been made in bone tissue engineering. In this regard, scaffolds have attracted a lot of attention as three dimensional substrates for cell attachment which improve successful tissue engineering. The aim of the present study was to provide an interconnected porous scaffold to facilitate cell infiltration. To do so, cancellous bone from bovine femur was dissected in fragments and decellularized by physicochemical methods, including snap freeze/thaw, rinsing in hot water and treatment with different solutions of sodium dodecyl sulfate (SDS). Histological analysis and 4',6-diamidino-2-phenylindole staining revealed that the best results were obtained after treatment with 2.5%, 5%, and 8% SDS for 8, 3, or 1 h respectively, which significantly removed bone cells with intact trabeculae geometry. Further characterization of decellularized scaffolds by the compression tests also revealed no significant difference between elastic modulus values of the three different SDS treatments. Moreover, studying the ratio of bone trabeculae to bone surfaces (BT/BS) as assessed by Clemex vision software 3.5 showed that treatment with 2.5% SDS for 8 h resulted in a BT/BS score in the range of native bone and therefore this treatment was used for further experiments. Histological studies and scanning electron microscopy revealed rat mesenchymal stem cells integration, adhesion, and maintenance during the 2 and 7 d of culture in vitro. In conclusion, the present results support the effective role of SDS in cancellous bovine bone decellularization and also propensity of treated samples in providing a suitable three-dimentional environment to support the maintenance and growth of mesenchymal stem cells. PMID:23708915

  2. High abundance of CD271+ multipotential stromal cells (MSCs) in intramedullary cavities of long bones

    PubMed Central

    Cox, George; Boxall, Sally A.; Giannoudis, Peter V.; Buckley, Conor T.; Roshdy, Tarek; Churchman, Sarah M.; McGonagle, Dennis; Jones, Elena

    2012-01-01

    Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed. Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34+ hematopoietic stem cells and CD31+ endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45?/low CD271+ MSC population and revealed a trend for higher MSC numbers (average 5 fold, n=6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73+CD105+CD90+) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM. Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a low-tech method of harvesting potentially large numbers of MSCs for regenerative therapies and research. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. PMID:21807134

  3. Bone cells, sclerostin, and FGF23: what's bred in the bone will come out in the flesh.

    PubMed

    Ott, Susan M

    2015-03-01

    Bone metabolism is linked to systemic diseases, and new research shows that the bone cells have endocrine functions that affect multiple organs. They secrete sclerostin, FGF23, prostaglandins, and osteocalcin. Pereira et al. examined gene expression of cells grown from bone biopsies of adolescents with renal osteodystrophy, as a first step to understanding how the bone-cell abnormalities contribute to cardiovascular and metabolic problems in these patients. PMID:25723633

  4. Bone marrow and bone marrow derived mononuclear stem cells therapy for the chronically ischemic myocardium

    SciTech Connect

    Waksman, Ron; Baffour, Richard

    2003-09-01

    Bone marrow stem cells have been shown to differentiate into various phenotypes including cardiomyocytes, vascular endothelial cells and smooth muscle. Bone marrow stem cells are mobilized and home in to areas of injured myocardium where they are involved in tissue repair. In addition, bone marrow secretes multiple growth factors, which are essential for angiogenesis and arteriogenesis. In some patients, these processes are not enough to avert clinical symptoms of ischemic disease. Therefore, in vivo administration of an adequate number of stem cells would be a significant therapeutic advance. Unfractionated bone marrow derived mononuclear stem cells, which contain both hematopoietic and nonhematopoietic cells may be more appropriate for cell therapy. Studies in animal models suggest that implantation of different types of stem cells improve angiogenesis and arteriogenesis, tissue perfusion as well as left ventricular function. Several unanswered questions remain. For example, the optimal delivery approach, dosage and timing of the administration of cell therapy as well as durability of improvements need to be studied. Early clinical studies have demonstrated safety and feasibility of various cell therapies in ischemic disease. Randomized, double blind and placebo-controlled clinical trials need to be completed to determine the effectiveness of stem cell.

  5. Osteoanagenesis after transplantation of bone marrow-derived mesenchymal stem cells using polyvinylidene chloride film as a scaffold.

    PubMed

    Hamajima, Soichiro; Hayashi, Tatsuhide; Sato, Yamato; Sasaki, Keisuke; Kawai, Tatsushi

    2011-01-01

    The aim of this study was to develop a new cell transplantation technique for osteoanagenesis at bone defect sites. Polyvinylidene chloride (PVDC) film was evaluated because of its good biocompatibility and flexibility. We used this film as both a cell scaffold and a barrier membrane. Initially, the cell compatibility of the PVDC film for fibroblast-like cells and osteoblast-like cells was confirmed. Subsequently, bone marrow cells were obtained from rats and cultured on PVDC films in two kinds of medium. The PVDC films with bone marrow-derived mesenchymal stem cells (MSCs) were then applied to critical-sized bone defects in the calvarial bone of rats. After the transplantation, the surgical sites were dissected out and evaluated by soft X-ray radiography, micro-CT analysis and histological examinations. The bone marrow-derived MSC-transplanted rats showed greater bone regeneration than the control rats. Therefore, PVDC film is considered to be useful as a scaffold for bone regeneration. PMID:21946492

  6. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging.

    PubMed

    Mindaye, Samuel T; Lo Surdo, Jessica; Bauer, Steven R; Alterman, Michail A

    2015-12-01

    Bone-marrow derived mesenchymal stromal cells (BMSCs) have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5], [6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results. PMID:26702413

  7. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    PubMed Central

    Mindaye, Samuel T.; Lo Surdo, Jessica; Bauer, Steven R.; Alterman, Michail A.

    2015-01-01

    Bone-marrow derived mesenchymal stromal cells (BMSCs) have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5], [6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results. PMID:26702413

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

    PubMed Central

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

    2014-01-01

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

  9. The impact of simulated and real microgravity on bone cells and mesenchymal stem cells.

    PubMed

    Ulbrich, Claudia; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Magnusson, Nils; Infanger, Manfred; Grosse, Jirka; Eilles, Christoph; Sundaresan, Alamelu; Grimm, Daniela

    2014-01-01

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

  10. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

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

  11. Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

    PubMed Central

    Sasaki, Jun-Ichi; Hashimoto, Masanori; Yamaguchi, Satoshi; Itoh, Yoshihiro; Yoshimoto, Itsumi; Matsumoto, Takuya; Imazato, Satoshi

    2015-01-01

    The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs. PMID:26047122

  12. Bone Impairment in Phenylketonuria Is Characterized by Circulating Osteoclast Precursors and Activated T Cell Increase

    PubMed Central

    Mussa, Alessandro; D'Amico, Lucia; Fiore, Ludovica; Garelli, Davide; Spada, Marco; Ferracini, Riccardo

    2010-01-01

    Background Phenylketonuria (PKU) is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. Methodology Peripheral blood mononuclear cell (PBMC) cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL). Flow cytometry was utilized to analyze osteoclast precursors (OCPs) and T cell phenotype. Tumour necrosis factor α (TNF-α), RANKL and osteoprotegerin (OPG) were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. Principal Findings Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS). This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. Conclusions Our results indicate that PKU spontaneous osteoclastogenesis depends on the circulating OCP increase and the activation of T cells. Osteoclastogenesis correlates with clinical parameters, suggesting its value as a diagnostic tool for an early assessment of an increased bone resorption in PKU patients. PMID:21152388

  13. Decreased IL-1 beta and TNF alpha secretion in long-term bone marrow culture supernatant from Fanconi's anaemia patients.

    PubMed

    de Cremoux, P; Gluckman, E; Podgorniak, M P; Menier, C; Thierry, D; Calvo, F; Socié, G

    1996-09-01

    Recent studies have shown that abnormalities of cytokine and lymphokine secretion are involved in the pathophysiology of Fanconi's anaemia (FA). In the present study, we quantified IL-1 beta, IL-1 receptor antagonist (IL-1Ra), IL-6 and TNF alpha protein levels in the supernatant of long-term cultures generated from BM cells of FA patients. Cell-free conditioned medium from long-term bone marrow culture was harvested every week at confluence and tested for interleukin secretion. IL-1 beta, IL-1Ra, TNF alpha and IL-6 protein secretion was assessed using immunoassays. IL-6 secretion was similar between controls and FA supernatants from wk 1 to wk 4. TNF alpha released from FA cells was consistently found at very low levels compared to control cells during the first 3 wk. Furthermore, secretion of IL-1 beta by cells from FA was always more than 2 standard deviations below the value of IL-1 beta found in normal donor cells from wk 1 to wk 4. In conclusion, in addition to a stem cell defect, a marked decrease in IL-1 beta and TNF alpha secretion may be one of the mechanisms leading to bone marrow failure in individuals with Fanconi's anaemia. PMID:8898923

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

    PubMed Central

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

    2014-01-01

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

  15. Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

    NASA Technical Reports Server (NTRS)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

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

  16. Connective Tissue Growth Factor Reporter Mice Label a Subpopulation of Mesenchymal Progenitor Cells that Reside in the Trabecular Bone Region

    PubMed Central

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

    2014-01-01

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

  17. Osteoblasts subjected to mechanical strain inhibit osteoclastic differentiation and bone resorption in a co-culture system.

    PubMed

    Li, Jianyu; Wan, Zongming; Liu, Hui; Li, Hao; Liu, Lu; Li, Ruixin; Guo, Yong; Chen, Wei; Zhang, Xinchang; Zhang, Xizheng

    2013-10-01

    Bone remodeling is strictly mediated by the coupled activities of osteoblasts and osteoclasts, which are responsible for bone formation and resorption, respectively. Although many papers have been published on the mechanical responses of osteoblasts and osteoclasts, little is known about their communication during mechanical loading. In this study, a novel co-culture system was first established using Transwell culture inserts; MC3T3-E1 cells were embedded in the lower compartment of the inserts, and RAW264.7 cells were co-cultured in the upper compartment. The MC3T3-E1 cells were subjected to a mechanical strain of 2500?? at 0.5Hz to investigate the effect of strain-loaded osteoblasts on co-cultured osteoclasts. The results showed that osteoblast-like cells were activated with an increase of alkaline phosphatase (ALP) activities. The strain-conditioned medium caused decreased activity of tartrate-resistant acid phosphatase and reduced the number of mature multinucleated osteoclasts, which subsequently resulted in the suppressed formation of resorption pits. The expression levels of cathepsin-K and matrix metalloproteinase-9 were also depressed by the strain-conditioned medium. In addition, we found that the expression ratio between osteoprotegerin (OPG) and receptor activator of NF-kB ligand in osteoblasts was significantly up-regulated due to the enhanced levels of OPG. In summary, we conclude that the strain-stimulated osteoblasts inhibited the differentiation and bone resorption of osteoclasts and that the mechanism was associated with the increased secretion of OPG in osteoblasts. PMID:23609024

  18. Stem cell origin differently affects bone tissue engineering strategies

    PubMed Central

    Mattioli-Belmonte, Monica; Teti, Gabriella; Salvatore, Viviana; Focaroli, Stefano; Orciani, Monia; Dicarlo, Manuela; Fini, Milena; Orsini, Giovanna; Di Primio, Roberto; Falconi, Mirella

    2015-01-01

    Bone tissue engineering approaches are encouraging for the improvement of conventional bone grafting technique drawbacks. Thanks to their self-renewal and multi-lineage differentiation ability, stem cells are one of the major actors in tissue engineering approaches, and among these adult mesenchymal stem cells (MSCs) hold a great promise for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs) are the first- identified and well-recognized stem cell population used in bone tissue engineering. Nevertheless, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The fruitful selection and combination of tissue engineered scaffold, progenitor cells, and physiologic signaling molecules allowed the surgeon to reconstruct the missing natural tissue. On the basis of these considerations, we analyzed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e., periodontal ligament, maxillary periosteum) as well as adipose-derived stem cells (ASCs), in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, taking into account their specific features, they could be intriguing cell sources in different stem cell-based bone/periodontal tissue regeneration approaches. PMID:26441682

  19. Stem cell origin differently affects bone tissue engineering strategies.

    PubMed

    Mattioli-Belmonte, Monica; Teti, Gabriella; Salvatore, Viviana; Focaroli, Stefano; Orciani, Monia; Dicarlo, Manuela; Fini, Milena; Orsini, Giovanna; Di Primio, Roberto; Falconi, Mirella

    2015-01-01

    Bone tissue engineering approaches are encouraging for the improvement of conventional bone grafting technique drawbacks. Thanks to their self-renewal and multi-lineage differentiation ability, stem cells are one of the major actors in tissue engineering approaches, and among these adult mesenchymal stem cells (MSCs) hold a great promise for regenerative medicine strategies. Bone marrow MSCs (BM-MSCs) are the first- identified and well-recognized stem cell population used in bone tissue engineering. Nevertheless, several factors hamper BM-MSC clinical application and subsequently, new stem cell sources have been investigated for these purposes. The fruitful selection and combination of tissue engineered scaffold, progenitor cells, and physiologic signaling molecules allowed the surgeon to reconstruct the missing natural tissue. On the basis of these considerations, we analyzed the capability of two different scaffolds, planned for osteochondral tissue regeneration, to modulate differentiation of adult stem cells of dissimilar local sources (i.e., periodontal ligament, maxillary periosteum) as well as adipose-derived stem cells (ASCs), in view of possible craniofacial tissue engineering strategies. We demonstrated that cells are differently committed toward the osteoblastic phenotype and therefore, taking into account their specific features, they could be intriguing cell sources in different stem cell-based bone/periodontal tissue regeneration approaches. PMID:26441682

  20. Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

    PubMed

    Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

    2014-05-01

    We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for in vitro bone engineering. PMID:24685477

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

    PubMed Central

    SHAH, AMITA R.; SHAH, SARITA R.; OH, SUNHO; ONG, JOO L.; WENKE, JOSEPH C.; AGRAWAL, C. MAULI

    2014-01-01

    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 exper iment, human umbilical vein endothelial cells (EC) and normal human primary osteoblasts (OB) were co-cultured to evaluate cell migration and interactions within this biphasic composite scaffold. Both cell types were able to migrate between the different material phases of the scaffold. It was also observed that OB migration increased when they were co-cultured with ECs, whereas EC migration decreased in co-culture. The results show that co-culture of ECs and OBs in this composite biphasic scaffold allows for migration of cells throughout the scaffold and that pre-seeding a scaffold with ECs can increase OB infiltration into desired areas of the scaffold. PMID:21769541

  2. The separation of a mixture of bone marrow stem cells from tumor cells: an essential step for autologous bone marrow transplantation

    SciTech Connect

    Rubin, P.; Wheeler, K.T.; Keng, P.C.; Gregory, P.K.; Croizat, H.

    1981-10-01

    KHT tumor cells were mixed with mouse bone marrow to simulate a sample of bone marrow containing metastatic tumor cells. This mixture was separated into a bone marrow fraction and a tumor cell fraction by centrifugal elutriation. Elutriation did not change the transplantability of the bone marrow stem cells as measured by a spleen colony assay and an in vitro erythroid burst forming unit assay. The tumorogenicity of the KHT cells was similarly unaffected by elutriation. The data showed that bone marrow cells could be purified to less than 1 tumor cell in more than 10/sup 6/ bone marrow cells. Therefore, purification of bone marrow removed prior to lethal radiation-drug combined therapy for subsequent autologous transplantation appears to be feasible using modifications of this method if similar physical differences between human metastatic tumor cells and human bone marrow cells exist. This possibility is presently being explored.

  3. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    PubMed

    Xia, Jing; Luo, Min; Ni, Ni; Chen, Junzhao; Hu, Yamin; Deng, Yuan; Ji, Jing; Zhou, Jibo; Fan, Xianqun; Gu, Ping

    2013-01-01

    During retina development, retinal progenitor cell (RPC) proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs) are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM) which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC) self-renewal, as well as betacellulin (BTC), an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs) and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro. PMID:24098776

  4. 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 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference...

  5. 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 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference...

  6. 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 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures....

  7. 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 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures....

  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 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures....

  9. AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM

    EPA Science Inventory

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

  10. Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D.

    PubMed

    Kleinhans, C; Schmid, F F; Schmid, F V; Kluger, P J

    2015-07-10

    Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVβ3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts. PMID:25562421

  11. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation.

    PubMed

    Brady, Robert T; O'Brien, Fergal J; Hoey, David A

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24 hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. PMID:25721667

  12. Cells Derived from Young Bone Marrow Alleviate Renal Aging

    PubMed Central

    Yang, Hai-Chun; Rossini, Michele; Ma, Li-Jun; Zuo, Yiqin; Ma, Ji

    2011-01-01

    Bone marrow-derived stem cells may modulate renal injury, but the effects may depend on the age of the stem cells. Here we investigated whether bone marrow from young mice attenuates renal aging in old mice. We radiated female 12-mo-old 129SvJ mice and reconstituted them with bone marrow cells (BMC) from either 8-wk-old (young-to-old) or 12-mo-old (old-to-old) male mice. Transfer of young BMC resulted in markedly decreased deposition of collagen IV in the mesangium and less β-galactosidase staining, an indicator of cell senescence. These changes paralleled reduced expression of plasminogen activator inhibitor-1 (PAI-1), PDGF-B (PDGF-B), the transdifferentiation marker fibroblast-specific protein-1 (FSP-1), and senescence-associated p16 and p21. Tubulointerstitial and glomerular cells derived from the transplanted BMC did not show β-galactosidase activity, but after 6 mo, there were more FSP-1-expressing bone marrow-derived cells in old-to-old mice compared with young-to-old mice. Young-to-old mice also exhibited higher expression of the anti-aging gene Klotho and less phosphorylation of IGF-1 receptor β. Taken together, these data suggest that young bone marrow-derived cells can alleviate renal aging in old mice. Direct parenchymal reconstitution by stem cells, paracrine effects from adjacent cells, and circulating anti-aging molecules may mediate the aging of the kidney. PMID:21965376

  13. Dendritic Cell-Mediated In Vivo Bone Resorption

    PubMed Central

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

    2013-01-01

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

  14. Treatment with at Homeopathic Complex Medication Modulates Mononuclear Bone Marrow Cell Differentiation

    PubMed Central

    Cesar, Beatriz; Abud, Ana Paula R.; de Oliveira, Carolina C.; Cardoso, Francolino; Bernardi, Raffaello Popa Di; Guimarães, Fernando S. F.; Gabardo, Juarez; de Freitas Buchi, Dorly

    2011-01-01

    A homeopathic complex medication (HCM), with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now report in vivo immunophenotyping of total bone marrow cells and ex vivo effects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measured in vitro. After in vivo treatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects of ex vivo HCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were induced in vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products. PMID:19736221

  15. Dynamic culture improves cell reprogramming efficiency.

    PubMed

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling. PMID:27031931

  16. Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis.

    PubMed

    Cheung, Laurence C; Strickland, Deborah H; Howlett, Meegan; Ford, Jette; Charles, Adrian K; Lyons, Karen M; Brigstock, David R; Goldschmeding, Roel; Cole, Catherine H; Alexander, Warren S; Kees, Ursula R

    2014-07-01

    Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis. PMID:24727816

  17. Effects of developmental exposure to perfluorooctanoic acid (PFOA) on long bone morphology and bone cell differentiation.

    PubMed

    Koskela, A; Finnilä, M A; Korkalainen, M; Spulber, S; Koponen, J; Håkansson, H; Tuukkanen, J; Viluksela, M

    2016-06-15

    Perfluorooctanoic acid (PFOA) is a ubiquitous and persistent environmental chemical, which has been used extensively due to its stability and surface tension-lowering properties. Toxicological effects include induction of neonatal mortality and reproductive toxicity. In this study, pregnant C57BL/6 mice were exposed orally to 0.3mg PFOA/kg/day throughout pregnancy, and female offspring were studied at the age of 13 or 17months. Morphometrical and biomechanical properties of femurs and tibias were analyzed with micro-computed tomography and 3-point bending, and bone PFOA concentrations were determined by mass spectrometry. The effects of PFOA on bone cell differentiation were studied in osteoclasts from C57BL/6 mice and in the MC3T3 pre-osteoblast cell line. PFOA exposed mice showed increased femoral periosteal area as well as decreased mineral density of tibias. Biomechanical properties of these bones were not affected. Bone PFOA concentrations were clearly elevated even at the age of 17months. In osteoblasts, low concentrations of PFOA increased osteocalcin (OCN) expression and calcium secretion, but at PFOA concentrations of 100μM and above osteocalcin (OCN) expression and calcium secretion were decreased. The number of osteoclasts was increased at all PFOA concentrations tested and resorption activity dose-dependently increased from 0.1-1.0μM, but decreased at higher concentrations. The results show that PFOA accumulates in bone and is present in bones until the old age. PFOA has the potential to influence bone turnover over a long period of time. Therefore bone is a target tissue for PFOA, and altered bone geometry and mineral density seem to persist throughout the life of the animal. PMID:27068293

  18. Replication of cultured lung epithelial cells

    SciTech Connect

    Guzowski, D.; Bienkowski, R.

    1986-03-05

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

  19. Histochemistry of blood and bone marrow cells in pangolins.

    PubMed

    Caxton-Martins, A E

    1977-04-01

    Blood and bone marrow cells of pangolins have been examined histochemically. Sudanophilia, PAS positivity and acid phosphatase and alkaline phosphatase reactivity were confined to cells of the granulocytic and monocytic series, while peroxidase reactivity was confined to cells of the erythroid series. In this latter respect the pangolin is unique among mammals so far studied. PMID:858692

  20. Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis.

    PubMed

    Sasaki, Soichiro; Baba, Tomohisa; Nishimura, Tatsunori; Hayakawa, Yoshihiro; Hashimoto, Shin-Ichi; Gotoh, Noriko; Mukaida, Naofumi

    2016-08-01

    From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity. PMID:27177471

  1. Long Term Maintenance of Myeloid Leukemic Stem Cells Cultured with Unrelated Human Mesenchymal Stromal Cells

    PubMed Central

    Ito, Sawa; Barrett, A. John; Dutra, Amalia; Pak, Evgenia; Miner, Samantha; Keyvanfar, Keyvan; Hensel, Nancy F.; Rezvani, Katayoun; Muranski, Pawel; Liu, Paul

    2015-01-01

    Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6 weeks. Four samples showed CD34+CD38− predominance, and four were predominantly CD34+CD38+. CD34+ CD38− predominant leukemia cells maintained the CD34+ CD38− phenotype and were viable for 6 weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34+ CD38+ predominant leukemic cells maintained the CD34+CD38+ phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4 weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34+ blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6 weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest MSCs provide pro-survival benefits to leukemic blasts through cell-cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs. PMID:25535865

  2. Good Caco-2 cell culture practices.

    PubMed

    Natoli, Manuela; Leoni, Bruno D; D'Agnano, Igea; Zucco, Flavia; Felsani, Armando

    2012-12-01

    The human Caco-2 cells differentiate spontaneously in culture forming monolayers of mature intestinal enterocytes which have been used as a model of the intestinal barrier for in vitro toxicology studies. Reproducibility problems often reported in literature have been generally ascribed to different culture-related conditions, such as the type of animal serum used, the supplements added to the culture media, the passage number and the source of cell clones. The Caco-2 cell culture protocol here described has been recently optimized in our laboratory, producing a homogeneous and highly polarized monolayer of cells which display many of the characteristics of the intestinal enterocytes. This protocol differs from standard protocols mainly because Caco-2 cells are subcultured when they reach just 50% of confluence, instead of 80%, retaining a high proliferation potential. When this cell population is seeded at high density on filter inserts differentiates almost synchronously and much more homogenously. PMID:22465559

  3. Good Caco-2 cell culture practices.

    TOXLINE Toxicology Bibliographic Information

    Natoli M; Leoni BD; D'Agnano I; Zucco F; Felsani A

    2012-12-01

    The human Caco-2 cells differentiate spontaneously in culture forming monolayers of mature intestinal enterocytes which have been used as a model of the intestinal barrier for in vitro toxicology studies. Reproducibility problems often reported in literature have been generally ascribed to different culture-related conditions, such as the type of animal serum used, the supplements added to the culture media, the passage number and the source of cell clones. The Caco-2 cell culture protocol here described has been recently optimized in our laboratory, producing a homogeneous and highly polarized monolayer of cells which display many of the characteristics of the intestinal enterocytes. This protocol differs from standard protocols mainly because Caco-2 cells are subcultured when they reach just 50% of confluence, instead of 80%, retaining a high proliferation potential. When this cell population is seeded at high density on filter inserts differentiates almost synchronously and much more homogenously.

  4. Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

    SciTech Connect

    Shima, Haruko; Takubo, Keiyo; Iwasaki, Hiroko; Yoshihara, Hiroki; Gomei, Yumiko; Hosokawa, Kentaro; Arai, Fumio; Takahashi, Takao; Suda, Toshio

    2009-01-16

    Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2R{gamma}{sup null} (NOG) mice. Hypoxic culture (1% O{sub 2}) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34{sup +}CD38{sup -} cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.

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

    PubMed

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

    2014-01-01

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

  6. Emulsions Containing Perfluorocarbon Support Cell Cultures

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

    Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting,