Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

Leboy, Phoebe S.; Beresford, J.N.; Devlin, C; Owen, Maureen

doi:10.1002/jcp.1041460306

Cited by 321 publications

(179 citation statements)

References 41 publications

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“…9,[11][12][13][14][15][16][17]22,29,[63][64][65][66][67] The main rationale behind the use of these supplements is to provide signaling proteins or enzymes to activate mineral production by MSCs. Previous in vitro 2D studies have shown that the direct coculture of MSCs with endothelial cells [36][37][38][39] or chondrocytes 68 can provide the necessary factors to induce early markers of osteogenic differentiation (ALP expression) without the need for these supplements.…”

Section: Discussionmentioning

confidence: 99%

“…[4][5][6][7][8] The standard procedure to induce osteogenic differentiation of MSCs in vitro is through the culture of the cells in the presence of a cocktail of dexamethasone, ascorbic acid, and b-glycerophosphate. [9][10][11][12][13][14][15][16][17] Dexamethasone is a steroid that causes MSC differentiation into osteoblasts by activation of the WNT/b-catenin signaling pathway, which in turn activates Runx2 expression and induces the differentiation of MSCs into immature osteoblasts. [18][19][20] Ascorbic acid acts as a cofactor for enzymes that hydroxylate proline and lysine in collagen 21 and participates in collagen chain formation.…”

mentioning

confidence: 99%

“…12,[23][24][25] It also regulates expression of genes including osteopontin and BMP-2. [26][27][28] Exposure of rat MSCs, 12,[14][15][16][17] human MSCs (hMSCs), 9,11,13 or murine osteoblasts 22,29 to dexamethasone, ascorbic acid, and b-glycerophosphate can significantly increase alkaline phosphatase (ALP) activity in vitro.…”

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confidence: 99%

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Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Freeman

Stevens

Owens

et al. 2016

Tissue Engineering Part A

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In vitro bone regeneration strategies that prime mesenchymal stem cells (MSCs) with chondrogenic factors, to mimic aspects of the endochondral ossification process, have been shown to promote mineralization and vascularization by MSCs both in vitro and when implanted in vivo. However, these approaches required the use of osteogenic supplements, namely dexamethasone, ascorbic acid, and b-glycerophosphate, none of which are endogenous mediators of bone formation in vivo. Rather MSCs, endothelial progenitor cells, and chondrocytes all reside in proximity within the cartilage template and might paracrineally regulate osteogenic differentiation. Thus, this study tests the hypothesis that an in vitro bone regeneration approach that mimics the cellular niche existing during endochondral ossification, through coculture of MSCs, endothelial cells, and chondrocytes, will obviate the need for extraneous osteogenic supplements and provide an alternative strategy to elicit osteogenic differentiation of MSCs and mineral production. The specific objectives of this study were to (1) mimic the cellular niche existing during endochondral ossification and (2) investigate whether osteogenic differentiation could be induced without the use of any external growth factors. To test the hypothesis, we evaluated the mineralization and vessel formation potential of (a) a novel methodology involving both chondrogenic priming and the coculture of human umbilical vein endothelial cells (HUVECs) and MSCs compared with (b) chondrogenic priming of MSCs alone, (c) addition of HUVECs to chondrogenically primed MSC aggregates, (d-f) the same experimental groups cultured in the presence of osteogenic supplements and (g) a noncoculture group cultured in the presence of osteogenic growth factors alone. Biochemical (DNA, alkaline phosphatase [ALP], calcium, CD31 + , vascular endothelial growth factor [VEGF]), histological (alcian blue, alizarin red), and immunohistological (CD31 + ) analyses were conducted to investigate osteogenic differentiation and vascularization at various time points (1, 2, and 3 weeks). The coculture methodology enhanced both osteogenesis and vasculogenesis compared with osteogenic differentiation alone, whereas osteogenic supplements inhibited the osteogenesis and vascularization (ALP, calcium, and VEGF) induced through coculture alone. Taken together, these results suggest that chondrogenic and vascular priming can obviate the need for osteogenic supplements to induce osteogenesis of human MSCs in vitro, while allowing for the formation of rudimentary vessels.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Freeman

Stevens

Owens

et al. 2016

Tissue Engineering Part A

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“…Glucocorticoid can stimulate, inhibit, or have a biphasic effect on collagen mRNA expression, depending on the osteoblast systems and culture conditions (Dietrich et al 1979, Kasugai et al 1991, Shalhoub et al 1992, Yao et al 1994. In rat BMSC, dexamethasone decreased the level of type I collagen mRNA during the first 6 or 7 days of treatment, followed by a rebound to near control on day 9 and, finally, an increase of four-to fivefold 14-28 days after treatment (Kasugai et al 1991, Leboy et al 1991, Yao et al 1994. Our results showed that the expression of type I collagen mRNA in hBMSC was decreased 1 day after treatment, and slightly rebounded in the next 7 days, although the level remained inhibited by dexamethasone over a 4-week period.…”

Section: Discussionmentioning

confidence: 99%

Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of glucocorticoid-induced bone loss

Sl²,

Gs³

1999

Journal of Endocrinology

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It is well documented that glucocorticoid excess causes bone loss, but the mechanisms of these effects remain poorly defined. To understand further the mechanisms of glucocorticoid-induced osteoporosis, we investigated the effects of glucocorticoids on bone formation and bone resorption by examining the proliferation, functional activities, and cytokine secretion of cultured human bone marrow stromal cells (hBMSC). Treatment with dexamethasone for 24 h at the concentration of 10 8 M significantly suppressed [ 3 H]thymidine incorporation and further inhibition was observed with longer treatment (8 days) or higher concentration (10 7 M). Alkaline phosphatase activity of hBMSC was markedly stimulated with addition of dexamethasone (10 8 M), to 191 22% (after 4 days) and 317 46% (after 7 days) of control. Dexamethasone (10 8 M) treatment for 48 h decreased the incorporation of [ 3 H]proline into collagenasedigestible protein (CDP; 43·7 7·9% of control) and non-collagen protein (65·2 8·4% of control), with a greater effect on CDP. Northern blot analysis indicated that 1(I)-collagen mRNA level was decreased by dexamethasone to 27·6 9·0% of the control value after 1 day of exposure, and to 55·2 6·2% after 7 days. Dexamethasone markedly suppressed basal production of interleukin (IL)-6 and IL-11 and that stimulated by parathyroid hormone (PTH), IL-1 , or tumour necrosis factor-in a dose-dependent manner. These results suggest that the glucocorticoid-induced bone loss is derived at least in part via inhibition of bone formation, which includes the suppression of osteoblast proliferation and collagen synthesis. As both basal and PTH-stimulated production of IL-6 and IL-11 are decreased by dexamethasone, the increased bone resorption observed in glucocorticoidinduced osteopenia does not appear to be mediated by IL-6 or IL-11.

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“…[1][2][3] Induction of osteoprogenitor differentiation of MSCs by dexamethasone (Dex) is well known, 4,5 and culture supplementation with Dex at 10 À8 M has been reported to promote osteoprogenitor cell differentiation in marrow stromal cells. 6,7 In addition, Dex is also a component of medium used for differentiation into chondroblasts, myocytes, and adipocytes in vitro. [8][9][10] With respect to Dex effects on apoptosis, the results have often been contradictory, depending upon the cell type being studied.…”

mentioning

confidence: 99%

Dexamethasone inhibition of confluence‐induced apoptosis in human mesenchymal stem cells

Song

Caplan

Dennis

2008

Journal Orthopaedic Research

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Human mesenchymal stem cells (MSCs) have been extensively characterized with respect to their in vitro expansion and differentiation potential, especially with respect to osteogenesis. Dexamethasone (Dex) is a well-known inducer of osteogenic differentiation of MSCs, but little is known about the effect of Dex treatment on apoptosis in MSCs. In this study, apoptosis in MSCs was examined with respect to cell density and Dex supplementation, using DAPI staining and DNA fragmentation ELISA Assay. In MSC cultures initiated at 1.0, 3.0, and 9.0 Â 10 3 cells per cm 2 , it was found that higher MSC density correlated with increased apoptosis and that this apoptotic effect was diminished in cultures containing 100 nM Dex. MSCs and fibroblasts were co-cultured, along with empty insert controls, and assayed for apoptosis by ELISA and DAPI counts to determine if soluble factors accounted for the cell density-related apoptosis. No difference was seen between MSCs cultured with inserts containing either MSCs, fibroblasts, or empty control. To determine cell contact effects, BrdU-labeled MSCs were cultured alone or with unlabeled chondrocytes at 2Â and 8Â the number of MSCs, with and without Dex, and apoptosis levels quantified. The results showed increased apoptosis at greater cell densities, and that the amount of apoptosis was greatly diminished in cultures containing Dex. These results show that apoptosis in MSCs is cell density-related, requires direct cell contact, and that Dex treatment reduces or eliminates this density-related apoptosis. These results may impact how MSCs should be cultured for clinical applications. Keywords: dexamethasone; mesenchymal stem cells; apoptosis Human bone marrow-derived mesenchymal stem cells (MSCs) are a rare population of undifferentiated cells that have the capacity to self-renew and can differentiate into mesodermal phenotypes including osteoblasts, chondrocytes, myocytes, and adipocytes. 1-3 Induction of osteoprogenitor differentiation of MSCs by dexamethasone (Dex) is well known, 4,5 and culture supplementation with Dex at 10 À8 M has been reported to promote osteoprogenitor cell differentiation in marrow stromal cells. 6,7 In addition, Dex is also a component of medium used for differentiation into chondroblasts, myocytes, and adipocytes in vitro. [8][9][10] With respect to Dex effects on apoptosis, the results have often been contradictory, depending upon the cell type being studied. For example, Dex treatment has been shown to induce apoptosis in thymocytes, 11,12 lymphocytes, 13-15 some tumor cells, 16,17 and respiratory epithelium, 18,19 whereas primary cultured hepatocytes, 20 neutrophils, 21,22 and some solid tumors 23,24 show reduced apoptosis after Dex treatment.Interestingly, while Dex has been used extensively with MSCs as an inductive factor for multiple end-stage phenotypes, few studies have assessed the effects of Dex on proliferation and apoptosis. To date, only one other study addressed the issue of Dex effects on MSC apoptosis wherein it was shown that hu...

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Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

Cited by 321 publications

References 41 publications

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of glucocorticoid-induced bone loss

Dexamethasone inhibition of confluence‐induced apoptosis in human mesenchymal stem cells

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