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Richard O C Oreffo - One of the best experts on this subject based on the ideXlab platform.
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the effect of the delivery of vascular endothelial growth factor and bone morphogenic protein 2 to Osteoprogenitor Cell populations on bone formation
Biomaterials, 2010Co-Authors: Janos M Kanczler, Patrick J Ginty, Lisa J White, N M P Clarke, Steven M Howdle, Kevin M Shakesheff, Richard O C OreffoAbstract:Regenerating bone tissue involves complex, temporal and coordinated signal cascades of which bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF165) play a prominent role. The aim of this study was to determine if the delivery of human bone marrow stromal Cells (HBMSC) seeded onto VEGF165/BMP-2 releasing composite scaffolds could enhance the bone regenerative capability in a critical sized femur defect. Alginate-VEGF165/PDLLA-BMP-2 scaffolds were fabricated using a supercritical CO2 mixing technique and an alginate entrapment protocol. Increased release of VEGF165 (750.4 ± 596.8 ?g/ml) compared to BMP-2 (136.9 ± 123.4 ?g/ml) was observed after 7-days in culture. Thereafter, up till 28 days, an increased rate of release of BMP-2 compared to VEGF165 was observed. The alginate-VEGF165/PDLLA-BMP-2 + HBMSC group showed a significant increase in the quantity of regenerated bone compared to the alginate-VEGF165/PDLLA-BMP-2 and alginate/PDLLA groups respectively in a critical sized femur defect study as indices measured by ?CT. Histological examination confirmed significant new endochondral bone matrix in the HBMSC seeded alginate-VEGF165/PDLLA-BMP-2 defect group in comparison to the other groups. These studies demonstrate the ability to deliver a combination of HBMSC with angiogenic and osteogenic factors released from biodegradable scaffold composites enhances the repair and regeneration of critical sized bone defects.
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differential in gel electrophoresis dige analysis of human bone marrow Osteoprogenitor Cell contact guidance
Acta Biomaterialia, 2009Co-Authors: Fahsai Kantawong, Richard Burchmore, C D W Wilkinson, Richard O C Oreffo, Matthew J DalbyAbstract:We have used a recent comparative proteomics technique, differential in-gel electrophoresis (DIGE), to study Osteoprogenitor Cell response to contact guidance in grooves. In order to increase protein output from small sample sizes, we used bioreactor culture before protein extraction and gel electrophoresis. Mass spectroscopy was used for protein identification. A number of distinct proteins were observed to exhibit significant changes in expression. These changes in protein expression suggest that the Cells respond to tailored grooved topographies, with alterations in their proteome concurrent with changes in Osteoprogenitor phenotype.
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natural marine sponge fiber skeleton a biomimetic scaffold for human Osteoprogenitor Cell attachment growth and differentiation
Tissue Engineering, 2003Co-Authors: David W Green, Daniel Howard, Xuebin Yang, M Kelly, Richard O C OreffoAbstract:Identification of suitable scaffolds onto which human stem Cells can be seeded to generate functional three-dimensional tissues is a major research goal. A natural marine sponge skeleton was selected as a potential scaffold on the basis of the hydration potential of the fiber, the presence of open interconnected channels created by the fiber network, the collagenous composition of the fiber, and the structural diversity of fiber architecture. The skeleton of an undetermined species of Spongia (Class Demospongiae: Order Dictyoceratida: Family Spongiidae), composed of spongin, supported growth of human Osteoprogenitor Cells. Cell attachment and invasion into the framework were observed within 16 h, followed by development into membranous sheets between the sponge fibers by concentric infilling. Histochemical staining for alkaline phosphatase and type I collagen indicated formation of bone matrix as confirmed by birefringence. At 9 and 14 days alkaline phosphatase-specific activity in sponge fiber-Osteoprogenitor Cell cultures was significantly greater than in control cultures on Cell culture plastic. Adsorption with recombinant human bone morphogenetic protein 2 confirmed the potential of this sponge skeleton as a delivery scaffold for osteogenic factors. The abundance and structural diversity of natural marine sponge skeletons and their potential as multifunctional, Cell conductive and inductive frameworks indicate a promising new source of scaffold for tissue regeneration.
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effects of interferon alpha on human Osteoprogenitor Cell growth and differentiation in vitro
Journal of Cellular Biochemistry, 1999Co-Authors: Richard O C Oreffo, C J Joyner, Silke Romberg, A S Virdi, Sigurd Berven, J T TriffittAbstract:The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic Cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating Cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), Cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow Osteoprogenitor Cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of Cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for Osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human Osteoprogenitor Cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local bone turnover through the specific and direct modulation of Osteoprogenitor proliferation and differentiation.
Jane E Aubin - One of the best experts on this subject based on the ideXlab platform.
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stanniocalcin 1 stimulates osteoblast differentiation in rat calvaria Cell cultures
Endocrinology, 2003Co-Authors: Yuji Yoshiko, Norihiko Maeda, Jane E AubinAbstract:Stanniocalcin 1 (STC1) is a mammalian homolog of STC, the fish calcium/phosphate-regulating polypeptide whose functions are only beginning to be elucidated. Recently, we demonstrated that STC1 stimulates, in an autocrine/paracrine fashion, bone mineralization by increasing phosphate uptake in osteoblasts apparently via the functional activity of the sodium-dependent phosphate transporter, Pit1. We have now assessed the role of STC1 on osteoblast development in fetal rat calvaria (RC) Cell cultures. STC1 mRNA and protein were differentially expressed over the time course of cultures, and dexamethasone, a potent stimulator of differentiation in this model, shifted peak STC1 expression levels to earlier times. Overexpression [recombinant human (rh) STC1] and underexpression (antisense oligonucleotides) of STC1 accelerated and retarded, respectively, osteogenic development as well as osteopontin and osteocalcin mRNA expression in mature osteoblast cultures, but not Osteoprogenitor Cell cultures. Dexamethasone shifted the effective doses required for these effects to higher and lower concentrations of antisense oligonucleotides and rhSTC1, respectively. Concomitantly, rhSTC1 increased both sodium-dependent phosphate uptake and Pit1 gene expression in nodule formation stages, but not in primitive progenitor stages of RC Cell cultures. Thus, STC1 accelerates osteoblast development in an autocrine/paracrine manner in the RC Cell culture model.
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Osteoprogenitor Cell differentiation to mature bone forming osteoblasts
Drug Development Research, 2000Co-Authors: Jane E Aubin, Johan N M HeerscheAbstract:Osteoblasts are the skeletal Cells responsible for synthesis, deposition and mineralization of the extraCellular matrix of bone. By mechanisms that are only beginning to be understood, stem Cells, primitive Osteoprogenitors and related mesenchymal precursors arise in the embryo where they participate in development of the skeleton; at least some persist in the adult organism, where they contribute to replacement of osteoblasts in bone turnover and in fracture healing. However, many questions remain as to the nature of these precursor Cell pools, including which Cells constitute a stem Cell pool vs. a committed progenitor pool, which pools persist in adult life and which constitute targets for hormones, cytokines, and growth factors during bone formation, turnover, and repair. During Osteoprogenitor proliferation and differentiation, a series of Cellular and molecular events occur that are characterized by sequential up- and downregulation of osteoblast-associated genes, including those for specific transcription factors, Cell cycle-related proteins, adhesion molecules, and matrix proteins; together these result in a mature matrix-synthesizing osteoblast. However, the mature osteoblast phenotype is itself heterogeneous, with subpopulations of osteoblasts expressing only subsets of the known osteoblast markers, raising intriguing questions, including how different pools of osteoblasts or their precursors may respond to therapeutic agents. Drug Dev. Res. 49:206–215, 2000. © 2000 Wiley-Liss, Inc.
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Osteoprogenitor Cell frequency in rat bone marrow stromal populations role for heterotypic Cell Cell interactions in osteoblast differentiation
Journal of Cellular Biochemistry, 1999Co-Authors: Jane E AubinAbstract:Glucocorticoids, notably dexamethasone (Dex), have been reported to be a requirement for Osteoprogenitor Cell differentiation in young adult rat bone marrow stromal Cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of Osteoprogenitor Cells present. Stromal Cells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase activity, hormone responsiveness, and matrix molecules, including type I collagen, osteopontin, bone sialoprotein, and osteocalcin), as well as their functional capacity to differentiate to form a mineralized bone nodule, were assessed. Dex increased, but was not an absolute requirement for, the expression of osteogenic markers. Bone nodule formation was plating Cell density dependent and occurred under all combinations of treatment with or without Dex but was maximal when Dex was present in both the primary and secondary cultures. Dex increased CFU-F by ∼2-fold, but increased CFU-O (Osteoprogenitor Cells; bone nodule forming Cells) by 5- to 50-fold depending on the Cell density and duration of treatment. Neither CFU-F nor CFU-O expression followed a linear relationship in limiting dilution analysis until very high Cell densities were reached, suggesting cooperativity of Cell types within the population and a multitarget phenomenon leading to Osteoprogenitor differentiation. When a large number of nonadherent bone marrow Cells or their conditioned medium was added to the stromal Cells, Osteoprogenitors comprised approximately 1/100 of plated adherent Cells and their expression followed a linear, single-hit relationship. By contrast, rat skin fibroblasts or their conditioned medium totally inhibited bone nodule formation. These data support the hypothesis that in marrow stroma, as in other bone Cell populations such as those from calvaria, there are at least two classes of Osteoprogenitor Cells: those differentiating in the absence of added glucocorticoid and those requiring glucocorticoid to differentiate, that more than one Cell type is limiting for stromal Osteoprogenitor differentiation suggesting a role for heterotypic Cell–Cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory Cells in the bone marrow to alter Osteoprogenitor Cell expression. J. Cell. Biochem. 72:396–410, 1999. © 1999 Wiley-Liss, Inc.
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tri iodothyronine t3 and dexamethasone interact to modulate Osteoprogenitor Cell differentiation in fetal rat calvaria Cell cultures
Bone, 1995Co-Authors: H Ishida, Jane E Aubin, C G Bellows, Johan N M HeerscheAbstract:Abstract We investigated the role of 3,5,3′-tri-iodothyronine (T3) in regulating differentiation of Osteoprogenitor Cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on Osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria Cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985–1992; 1987). In standard culture medium containing 15% fetal bovine serum (FBS), low concentrations of T3 (0.001 – 0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1–100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T3 markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T3 was present prior to confluency. Higher concentrations of T3 (1–100 nM) decreased nodule number whether or not Dex was added. We then cultured Cells in medium containing FBS from which T3 and T4 were removed by treatment with AG-1χ-10 resin. In both + or - Dex conditions, bone nodule formation was increased 1.5 to 2-fold in T3, T4-depleted medium when compared with cultures maintained in standard culture medium. In T3, T4- depleted medium, Dex increased bone nodule formation dose dependently with maximum effect at 10 nM Dex, essentially similar to the effects of Dex in standard medium. Similar to the T3 response in media containing nondepleted FBS, addition of T3 to cultures in medium containing T3, T4-depleted FBS decreased the number of nodules in a dose-dependent manner. These results show that in standard culture medium, T3 inhibits Osteoprogenitor Cell differentiation in the absence of Dex, but that in the presence of an optimal concentration of Dex, low concentrations of T3 stimulate Osteoprogenitor Cell differentiation. Also, the results of the experiments where Dex was added to T3, T4-depleted medium suggest that inhibitory factors other than T3, T4 may have been removed in the depleted serum.
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t3 and dexamethasone interact to modulate Osteoprogenitor Cell differentiation in fetal rat calvaria Cell cultures
1995Co-Authors: H Ishida, Jane E Aubin, C G Bellows, Johan N M HeerscheAbstract:We investigated the role of 3,5,3'-tri-iodothyronine (T3) in regulating differentiation of Osteoprogenitor Cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on Osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria Cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985-1992; 1987). In standard culture medium containing 15% fetal bovine serum (FBS), low concentrations of T a (0.001-0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1-100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T a markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T 3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T 3 was present prior to confluency. Higher concentrations of T a (1-100 nM) decreased nodule number whether or not Dex was added. We then cultured Cells in medium containing FBS from which T 3 and T 4 were removed by treatment with AG-1x-10 resin. In both + or - Dex conditions, bone nodule formation was increased 1.5 to 2-fold in T 3, T4-depleted medium when compared with cultures maintained in standard culture medium. In T3, T4-depleted medium, Dex increased bone nodule formation dose dependently with maximum effect at 10 nM Dex, essentially similar to the effects of Dex in standard medium. Similar to the T 3 response in media containing nondepleted FBS, addition of T 3 to cultures in medium containing T3, T4-depleted FBS decreased the number of nodules in a dosedependent manner. These results show that in standard culture medium, T 3 inhibits Osteoprogenitor Cell differentiation in the absence of Dex, but that in the presence of an optimal concentration of Dex, low concentrations of T a stimulate Osteoprogenitor Cell differentiation. Also, the results of the experiments where Dex was added to T3, T4-depleted medium suggest that inhibitory factors other than T3, T 4 may have been removed in the depleted serum. (Bone 16:545-549; 1995)
David Segal - One of the best experts on this subject based on the ideXlab platform.
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induction of Osteoprogenitor Cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal Cells
Journal of Cellular Biochemistry, 1993Co-Authors: Benjamin Y Klein, I Gal, Zipora Hartshtark, David SegalAbstract:Bone marrow stromal Cells contain colony forming Cells with the potential to differentiate into Osteoprogenitor (OPC) Cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and beta-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal Cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3-4 and 10-11. Between days 5 and 10 there was a drop in Rho retention/Cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 micrograms/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 micrograms/ml Rho for 12 days only slightly increased day 12 ALP activity/Cell, had no effect on day-21 mineralization and only the high dose, 10.0 micrograms/ml, doubled stromal Cell proliferation. Under 24 h incubation Rho concentrations of 1.0 microgram/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal Cells. The results indicate that under both culture conditions stromal Cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.
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selection of malonate resistant stromal Cell derived Osteoprogenitor Cells in vitro
Journal of Cellular Biochemistry, 1993Co-Authors: Benjamin Y Klein, I Gal, David SegalAbstract:Bone marrow stromal Cells give rise to Osteoprogenitor Cell (OPC) colonies, with characteristic mineralized bone nodules in vitro. During differentiation, OPCs in the culture are surrounded by heterogeneous populations of various Cell lineages and by different OPC differentiation stages. In the present study, attempts were made to increase the homogeneity of OPCs in culture. The reliance on energy metabolism restricted to glycolysis, which is specific to the premineralizing skeletal Cells, was tested as a selectable marker for Cells in this stage. Day 12 alkaline phosphatase (ALP) and day 20–21 calcium precipitates were used as early and late OPC differentiation markers. Malonate, a competitive inhibitor of succinate dehydrogenase, was added to the OPC stimulation medium, to interfere with the Krebs cycle-dependent energy metabolism operating in most of the stromal Cells. OPCs that entered the stage of energy metabolism restricted to glycolysis were expected to become malonate resistant. Malonate showed dose and time dependence, 10 mM malonate added on day 3, decreased day 12 ALP activity/well to the lowest level. Variations in time and length of exposure to malonate used during the first 12 days of differentiation showed an inverse correlation between specific ALP activity and Cell yield. Malonate-treated variations of specific ALP and of Cell yield indices were up to 30- to 40-fold larger than variations within day 21 calcium precipitates. Thus, calcifying Cells were almost unchanged relatively to noncalcifying Cells. These results indicate that malonate-resistant Cells are mostly selected, rather than induced, to differentiate by malonate. The results also show that stromal derived OPCs undergo a similar biochemical stage as in chondrocytes. © 1993 Wiley-Liss, Inc.
Joelle Amedee - One of the best experts on this subject based on the ideXlab platform.
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additive effect of rgd coating to functionalized titanium surfaces on human Osteoprogenitor Cell adhesion and spreading
Tissue Engineering Part A, 2008Co-Authors: Delphine Le Guilloubuffello, Reine Bareille, M Gindre, Andreas Sewing, Pascal Laugier, Joelle AmedeeAbstract:Titanium-based biomaterials for endosseous implants have found widespread applications in the orthopedic, maxillofacial, and dental domains. Indeed, the surface characteristics such as their chemical modification control considerably the Cellular response and, subsequently, the quality and the quantity of new-formed bone around the implant. In this study, human Osteoprogenitor (HOP) Cell adhesion on different titanium surfaces functionalized with hydroxyapatite (HA), type I collagen, or Arg-Gly-Asp (RGD)-containing peptides is investigated by the quartz crystal resonators and by confocal laser scanning microscopy (CLSM) for the imaging of focal contact formation. Data obtained by quartz crystal resonator technique revealed that RGD-containing peptides alone increase HOP Cell adhesion in early time period of culture. Moreover, association of RGD-containing peptides with either type I collagen or with HA layers induces an additive effect on HOP Cell adhesion compared to Ti-Coll or Ti-HA. CLSM shows both the area of focal contact by Cell unit and the cytoskeleton network organization to differ according to the surfaces. Interestingly, association of RGD-containing peptides with HA layers induces an additive effect on focal contact formation on HOP Cells compared to Ti-HA alone. These data confirm that an RGD peptide effect occurs in the early time of culture, which is beneficial for osteoblast to spreading, differentiation, and survival.
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effect of huvec on human Osteoprogenitor Cell differentiation needs heterotypic gap junction communication
American Journal of Physiology-cell Physiology, 2002Co-Authors: F Villars, Bertrand Guillotin, Reine Bareille, T Amedee, S Dutoya, Laurence Bordenave, Joelle AmedeeAbstract:Bone development and remodeling depend on complex interactions between bone-forming osteoblasts and other Cells present within the bone microenvironment, particularly vascular endothelial Cells tha...
Benjamin Y Klein - One of the best experts on this subject based on the ideXlab platform.
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induction of bone formation in rat Osteoprogenitor Cell culture by sera of climacteric women before and after hormone replacement therapy
Fertility and Sterility, 1999Co-Authors: Nathan Rojansky, Hannah Benbassat, Alina Mariash, Benjamin Y KleinAbstract:Abstract Objective: To evaluate the effect of hormone replacement therapy (HRT) on growth and differentiation of cultured Osteoprogenitor Cells. Design: Prospective clinical study. Setting: Outpatients in a menopause clinic. Patient(s): Women with climacteric symptoms. Intervention(s): Daily oral conjugated estrogen, 0.625 mg, and medroxyprogesterone acetate, 2.5 mg, for 7–12 months. Bone density measurement before HRT and blood sampling before and after HRT. Main Outcome Measure(s): Sera of climacteric women were added to the culture of rat Osteoprogenitor Cells, and indices of Cell proliferation and differentiation (alkaline phosphatase activity and mineralization) were measured before and after HRT. Result(s): Sera after HRT significantly decreased Cell counts but not alkaline phosphatase activity or mineralization as compared with sera before HRT. However, mineralization induced in the bioassay by both sera showed a positive correlation ( r = 0.56) with E 2 levels before treatment and a negative correlation ( r = −0.6181) with time in menopause of serum donors. The change in mineralization showed a significant correlation with hip bone mineral density z scores ( r = −0.67) but not with spine z scores ( r = −0.1915), whereas the change in Cell count correlated with spine bone mineral density z scores ( r = 0.49) only. Conclusion(s): Changes in serum-induced Cell proliferation and mineralization may be helpful in studying the response to HRT in climacteric women. Serum-induced mineralization is more efficient in diagnosing osteopenia than in monitoring HRT effects.
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induction of Osteoprogenitor Cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal Cells
Journal of Cellular Biochemistry, 1993Co-Authors: Benjamin Y Klein, I Gal, Zipora Hartshtark, David SegalAbstract:Bone marrow stromal Cells contain colony forming Cells with the potential to differentiate into Osteoprogenitor (OPC) Cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and beta-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal Cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3-4 and 10-11. Between days 5 and 10 there was a drop in Rho retention/Cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 micrograms/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 micrograms/ml Rho for 12 days only slightly increased day 12 ALP activity/Cell, had no effect on day-21 mineralization and only the high dose, 10.0 micrograms/ml, doubled stromal Cell proliferation. Under 24 h incubation Rho concentrations of 1.0 microgram/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal Cells. The results indicate that under both culture conditions stromal Cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.
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selection of malonate resistant stromal Cell derived Osteoprogenitor Cells in vitro
Journal of Cellular Biochemistry, 1993Co-Authors: Benjamin Y Klein, I Gal, David SegalAbstract:Bone marrow stromal Cells give rise to Osteoprogenitor Cell (OPC) colonies, with characteristic mineralized bone nodules in vitro. During differentiation, OPCs in the culture are surrounded by heterogeneous populations of various Cell lineages and by different OPC differentiation stages. In the present study, attempts were made to increase the homogeneity of OPCs in culture. The reliance on energy metabolism restricted to glycolysis, which is specific to the premineralizing skeletal Cells, was tested as a selectable marker for Cells in this stage. Day 12 alkaline phosphatase (ALP) and day 20–21 calcium precipitates were used as early and late OPC differentiation markers. Malonate, a competitive inhibitor of succinate dehydrogenase, was added to the OPC stimulation medium, to interfere with the Krebs cycle-dependent energy metabolism operating in most of the stromal Cells. OPCs that entered the stage of energy metabolism restricted to glycolysis were expected to become malonate resistant. Malonate showed dose and time dependence, 10 mM malonate added on day 3, decreased day 12 ALP activity/well to the lowest level. Variations in time and length of exposure to malonate used during the first 12 days of differentiation showed an inverse correlation between specific ALP activity and Cell yield. Malonate-treated variations of specific ALP and of Cell yield indices were up to 30- to 40-fold larger than variations within day 21 calcium precipitates. Thus, calcifying Cells were almost unchanged relatively to noncalcifying Cells. These results indicate that malonate-resistant Cells are mostly selected, rather than induced, to differentiate by malonate. The results also show that stromal derived OPCs undergo a similar biochemical stage as in chondrocytes. © 1993 Wiley-Liss, Inc.
