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  • 1
    ISSN: 1432-0827
    Keywords: Key words: Ectopic bone formation — Marrow osteoblasts — Cell differentiation — Histomorphometry.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. MBA-15.4 and MBA-15.6 cell lines are marrow stromal clonal subpopulations and represent various stages of differentiation of the osteoblastic family. These cells vary in terms of morphology, proliferation rate, synthesis of matrix proteins as collagen and noncollagenous proteins, and by their responses to hormones and growth factors. Their differential properties directly reflect the clonal cells' ability to form bone in vivo. When the cells were transplanted at an ectopic site, under the kidney capsule, MBA-15.4 line formed small foci of bone whereas MBA-15.6 cell line formed massive woven bone during the same period of time. In this study, we focused on the histomorphometric analysis of ectopic ossicles formed by the clonal cell lines. Assessments of bone mass changes involved measurements of cellular components, osteoid, and formation of primary bone. The bony tissue formed was condensed, no hemopoiesis was noted, and the ossicle was not remodeled. The histology studies were used for quantitative analysis of the ossicle formation and describe the dynamics of ossicles formed by the individual cell types.
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  • 2
    ISSN: 1432-0827
    Keywords: Marrow stroma ; Stromal cell lines ; Osteoblasts ; Cytokines ; MBA-15
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary Osteoblasts, members of the marrow stromal cellular network, may play an active role in the hemopoietic microenvironment as well as in bone remodeling. In this study, we examined the extent to which marrow-derived osteogenic cells (MBA-15) possess various stromal functions. This marrow stromal-derived cell line was shown by us to exhibit osteoblastic characteristics in culture and to form bone in vivo. These cells are shown here to constitutively produce and secrete cytokines identified as M-CSF, GM-CSF, and IL-6. MBA-15 cells modulate growth of normal and malignant myeloid and lymphoid cells as well as leukemia cell lines in vitro. Cell-cell interactions were studied in co-cultures with adherent MBA-15 cells and the target hemopoietic cells. Growth inhibition effects, observed under various experimental conditions, can be attributed to the presence of different soluble and membrane-bound inhibitory activities produced by MBA-15 cells. Thus, MBA-15 cells spontaneously produce both stimulators and inhibitors that can affect myeloid and lymphoid cell growth. Marrow osteogenic cells may therefore participate in the stromal regulation of hemopoiesis.
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  • 3
    ISSN: 1432-0827
    Keywords: Mineralization ; In vitro ; Stromal osteoblasts ; 3-D culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract The present study describes a new three-dimensional (3-D) culture system that enables the maintenance and phenotypic expression of bone marrow stromal osteoblasts. This culture substratum is advantageous in that it provides suitable conditions for attachment, growth, and differentiation of cells forming 3-D layers. The MBA-15 cell line was grown in unlimited quantities on 3-D Fibro-Cel carriers. These cells mineralized when exposed to ascorbic acid and β-glycerophosphate (βGP). Under these mineralization conditions, mRNA expressions of procollagen α2(I) and [3H]-proline-labeled protein were increased. The expression of mRNA for osteonectin and to a lesser extent, for osteopontin was increased, whereas alkaline phosphatase and biglycan remained unaffected under similar conditions. Exposure of mineralizing cultures to dexamethasone reduced mRNA of procollagen α2(I) and osteonectin to control level. Scanning electron microscopy revealed that cells were grown along the fabric's fibers and produced collagen fiberils. Under appropriate conditions, extensive mineralization had taken place. The mineralization process involves the formation of calcospherites, and correlates with an increase in calcium content. The Fibro-Cel carriers enable formation of 3-D architecture and mineralized tissue in vitro.
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  • 4
    ISSN: 0730-2312
    Keywords: stromal cells ; osteoblasts ; attachment ; bone matrix ; bone formation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this study, we report on the cell adhesion properties of marrow stromal cells to extracellular matrix components such as collagen and noncollagenous proteins. The osteoblastic cells and their non-osteoblastic counter-parts (MBA series) from the marrow stroma differentially recognized a spectrum of extracellular matrix proteins. The osteoblastic cells, MBA-15, preferentially attached to bone matrix proteins, whereas fibroendothelial MBA-2.1 and adipocytic 14F1.1 cells did not. The MBA-15 cells demonstrated a preference in their attachment to fibronectin 〉 mixture of collagens 〉 bone matrix extracts 〉 collagen type 1 〉 noncollagenous proteins. Clonal subpopulations derived from the MBA-15 cell line representing various stages along the osteogenic lineage expressed differential attachment preference. MBA-15.4, a less differentiated clonal line, was compared to MBA-15.6, a mature cell line. © 1995 Wiley-Liss, Inc.
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  • 5
    ISSN: 0730-2312
    Keywords: marrow stromal cells ; cell morphogenesis ; attachment ; ECM ; mRNA expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Our aim was to study the role of various extracellular matrices (ECM) on growth and differentiation of marrow stromal cells in vitro. Morphology changes, gene expression, and enzymatic activities were monitored in stromal osteoblastic MBA-15 and adipocytic 14F1.1 cells. These stromal cells were plated on dishes precoated with different substrata, such as matrigel (basement membrane), collagen type I, and endothelial ECM, and compared with cells plated on protein-free dishes. Striking morphological differences were observed when the cells grew on these different substrata. Changes in cell shape and growth also led to differential mRNA expression and enzymatic activities. When MBA-15 cells were plated on collagen, there was a decrease in mRNA for alkaline phosphatase (ALK-P), osteopontin (OP), and osteonectin (ON), and an increase in mRNA for procollagen (I). A differential effect was noted on 14F1.1 cells, the mRNA for ALK-P increased, the expressions of OP and ON lowered, and no expression for procollagen (I) was monitored. MBA-15 cells cultured on matrigel had decreased mRNA for ALK-P and OP, while they had increased ON mRNA expression and remained unchanged for procollagen 1. No change in mRNA expression by 14F1.1 cells was monitored when cultured on matrigel. Functional enzymatic activities of ALK-P markedly decreased in MBA-15 cells cultured on various substrata, and increased or were unchanged in 14F1.1 cells. An additional enzyme, neutral endopeptidase (CD10/NEP), altered differentially in both cell types; this enzymatic activity increased or was unchanged when cells were cultured on these matrices. The results indicate a specific role for different ECM on various stromal cell types and their function. © 1996 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 81-86 
    ISSN: 0730-2312
    Keywords: cell communication ; osteoblasts ; stromal cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We characterized the formation and regulation of the gap junction in calvarial osteoblasts and in a series of subtypes from marrow stromal cells. The stromal cells included osteogenic, chondro-osteogenic, and endothelial cells. The cell coupling was measured by using fluorescence dye injected into single cells, and its migration to neighboring cells was measured. The functional coupling of cells was highly expressed by the osteoblastic cells. This process is mediated through fast changes in intracellular Ca+2 levels. Calcium ionophore (A 23187) demonstrated an uncoupling effect on the cells. In addition, the exposure of the cells to the parathyroid hormone increased the formation of the gap junction complex; the highest level was demonstrated in the osteoblastic cells. J. Cell. Biochem. 69:81-86, 1998. © 1998 Wiley-Liss, Inc.
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  • 7
    ISSN: 0730-2312
    Keywords: vitamin A ; growth factors ; marrow stromal osteoblasts ; bone matrix proteins ; CD10/NEP ; neutral endopeptidase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effects of retinoic acid (RA) on the expression of osteoblastic-related cell makers was examined. A marrow and osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constituentively express mRNA encoding for procolllagen a2 (1), osteonectin, osteopontin, biglycan and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity dispalyed a different pattern. MBA-15.4, a presosteoblast cell ine, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor β (TGFβ). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic increase in MBA-15.6 cell responses to PTH and PGE2 but no significant effects could be observed in other clonal lines.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 69 (1998), S. 282-290 
    ISSN: 0730-2312
    Keywords: estrogen modulation ; osteoblastic cells ; plasma membrane receptors ; nuclear receptors ; gap junction communication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Two osteoblastic cell populations, calvarial and marrow stromal cells, were exposed to estrogen derivatives in vitro. The hormonal effect was monitored by following intracellular Ca+2 levels [Ca+2]i and gap-junction communication. We measured fast changes in intracellular Ca+2 levels in response, of these cells, to the steroid hormones. The changes were dose dependent revealing maximal activity at 100 pM by 17-β-Estradiol and 1 nM by estradiol-CMO. Additionally, the effect of estrogen, on functional coupling of the cells, was measured using fluorescence dye migration and counting the number of neighboring cells coupled by gap junctions. An uncoupling effect was demonstrated in response of these cells to estrogen treatment. The quick stereospecific effect was achieved in the presence of 17-β-estradiol but not in the presence of 17-α-estradiol. These results suggest the involvement of plasma membrane receptors in addition to the already known nuclear receptors in transducing the hormone effects in the osteoblastic cells. J. Cell. Biochem. 69:282-290, 1998. © 1998 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 62 (1996), S. 476-483 
    ISSN: 0730-2312
    Keywords: stromal osteoblasts ; dexamethasone ; attachment ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The clonal subtypes of cells in the osteogenic family represented by fibroblastoid MBA-15.33, preosteoblast MBA-15.4, and mature osteoblastic MBA-15.6 cells were used to study the effects of glucocorticoid (dexamethasone). The role of dexamethasone was monitored on cell attachment when plated on various protein substrata (BSA, collagen I, and Matrigel). A 24 h exposure of the cells to 10-6 M or 10-7 M dexamethasone differential affects their attachment preference. MBA-15.33 and MBA-15.4 cells increased their attachment capability on collagen I, while MBA-15.6 cells' attachment was inhibited. Pretreatment with (10-6 M) dexamethasone caused an increase in attachment on Matrigel by MBA-15.33 cells and to less extent by MBA-15.4 cells. Additionally, measurements of two enzymatic activities were monitored; one is alkaline phosphatase (ALK-P), and the second is neutral endopeptidase (CD10/NEP). MBA-15.33, MBA-15.4, and MBA-15.6 cells were exposed to dexamethasone or to various growth factors (bone morphogenic protein (BMP-2 and BMP-3), TGFβ, and IGF-I). In some experiments, pretreatment of cells by dexamethasone was followed by exposure to the growth factors. The cells' challenged cellular responses were not uniform and revealed a differential pattern when their ALK-P and CD10/NEP enzymatic activities were measured. © 1996 Wiley-Liss, Inc.
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  • 10
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This report documents characterization of five osteogenic cell subpopulations of bone marrow stroma. The clonally derived cell lines were isolated from the parental line MBA-15 known to express osteoblastic-associated features in vitro and to form bone in vivo. The latter, presumably “arrested” at a particular stage along the osteogenic lineage, are useful models to study the processes involved in the differentiation of bone forming cells. The clones differ in their morphology, proliferation rate, quantities and distribution of extracellular matrix proteins, levels of alkaline phosphatase activity and activation of adenylate cyclase by parathyroid hormone and/or prostaglandin E. These properties have been retained during prolonged growth and subculturing through many passages. MBA-15.4 is a presumptive preosteoblast with a fibroblast-like appearance; it proliferates rapidly, synthesizes equal amounts of collagen and noncollagenous proteins, and produces constitutively low levels of alkaline phosphatase. This clone has PGE2-stimulated adenylate cyclase activity and a very low constitutive response to PTH. On the other hand, MBA-15.6 has a large polygonal morphology with limited proliferative potential, synthesizes twice as much noncollagenous proteins as collagen, has high alkaline phosphatase activity, and responds strongly to PTH. The characteristics of the other clones place them between these two categories. The effects of 10-7 M dexamethasone or 10-12-10-8 M 1,25 dihydroxyvitamin D3 on growth and differentiation further strengthen the variance between these clones. The different in vitro characteristics of the various clones were directly reflected in their bone formation ability in vivo. When transplanted under the renal capsule, MBA-15.33 formed a thick fibrous tissue, MBA-15.4 formed small foci of bone, and MBA-15.6 formed massive woven bone at the same period of time. © 1993 Wiley-Liss, Inc.
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