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P5. In vitro properties of two immortalised stromal cell lines derived from rabbit bone marrow
Abstracts from the Summer Meeting, July 1993 because of its biocompatibility, very good mechanical properties and resorbability. We therefore studied the growth and differentiation of human bone marrow cells grown on non porous calcium carbonate. Human bone marrow cells were harvested from a 57 year old patient and cultured in alpha-MEM plus 1.5% FBS in four different conditions. 1: no addition, 2: 10-s M lp(OH)&, 3: IO-EM Dex, 4: lo-8 M Dex and 1,25(0H)D~. Resul*r: between day 5 and 20, DNA content was increased by a factor of 1.5 to 2 except in the presence of Dex and lfS(OH)D where it remained unchanged. Cells expressed a significant increase in alkaline phosphatase activity when they were cultured on calciulm carbonate only in the presence of dexamethasone and 1,25(OH)D3. Cells did not express any significant amount of alkaline phosphatase under the other conditions used. We conclude that lhuman bone marrow cells cultured on calcium carbonate in presence of Dex or l,XOHD3 are able to multiply and express alkaline phosphatase activity. However, no bone nodule formation with mineralisation was observed under these conditions. Aknowledgement: Herve Petite is in receipt of a Wellcome Trust Fellowship during the course of his work.
231 genes typical or specific for osteogenesis including the parathyroid hormone receptor alkaline phosphatase, osteopontin, osteonectin and osteocalcin. In addition to osteoblast-chondrocyte formation also differentiation into adipocytes is observed suggesting potential common progenitors for osteogenesis and adipogenesis. We investigated whether PTH in this system directs the differentiation into the osteogenic and/or other chondrogenic lineage. After expression of the rat PTH-receptor in BMP-~ansfected c3HlOT1/2 c&s adipogenesis is inhibited after addition of the liaand PTH (l-34). In addition surprisingly PTH (I-34) alone is s:fficient to induce nodul&ke structures in PTH-receptor transfected C3HlOT1/2 cells in the absence of BMP. Mineralisation was studied in bone marrow strornal cells. Element distribution and electron energy loss spectra images of phosphorus, calcium and oxygen show the localization of these elements within the same area indicating a chemical composition comparable to hydroxyapatite.
P5. In oitro properties of two immortalised rtromal cell lines derived from rabbit bone marrow E Brown, C Simpson, ME Nuttall*, B Ashton”, D Johnstone VIM Research Departmenf, Zeneca Pharmaceuticals, Mereside, Alderley Park Macclesfield Cheshire SK10 4TG; lSKB Phanmaceuticals, King of Prussia, PA. USA and **Department of
P3. Endothclial cells up-regulate alkaline phosphatarc expression by marrow stromal cells in co-culture RP Allsopp, AH Dnmunond*, LA N&ham*, JT Triffit
Rheumatology, Roberf Iones and Agnes Hospital, Oswestry, Shropshire SYlO 7AG
There is increasing interest in the role of endothelial cells in bone formatlon: the &se proximity of blood vessels to sites of active osteogenesis may reflect more than a requirement for a blood supply. Human marrow stromal (HM) cells were grown in co-culture for 5 days with bovine adrenal capillary endothelial (BACE) cells in DMIEM containing 10% foetal bovine serum, acidic fibroblast growth factor (long/ml) and heparin (15OOunits/ml). Histochemical and quantitative assays detected an increase in the HM-expression of alkaline phosphatase relative to that seen in the co-culture of HM and human skin cells or the culture of HM alone. If the BACE cells and HM cells were separated by a tissue culture Insert then no increase in alkaline phosphatase expression was seen. It is considered that HM cell populations, contain osteoprogenitors and alkaline phosphatase expression is an early marker of the osteogenic lineage. Our results suggest that endothelial cells are involved in specific interactions with HM cells causing differentiation towards the osteogenic phenotype. Further, this phenomenon requires cell-cell contact and is not solely mediated by the diffusion of soluble factors. Future work will investigate the mechanisms by which the two cell types interact.
Two cell clones, RCl and RC2, have been established by electroporation of rabbit bone marrow cells with a plasmid containing the temperature sensitive sv40 large T antigen and neomycin resistance genes (tsA58U19). RC2, but not RCI, has been shown to form bone in tivo; however, neither clone expressed significant levels of alkaline phosphatase (alk phos) in vitro even after dexamethasone or vitamin l& treatment (1). We have examined the effect of rhTGFg, rhEGF.and vitamin A on RCl and RC2 cell proliferation (measured bv thvmidine incorporation), and c;?llular differentiation (mea&red l& alk phos activity) in vitro, as well as examining their capacity to produce mineralised matrix when co-cultured with Cytodex beads (2). TGF8 (4-4OOpM) did not have any effect on RCl and RC2 aIk phos activity, bit inhibited cell proliferation in both clones at Sn&ml(~cO.OS>. EGF (0.1-1OOnM) caused an inhibition of alk ph& ac&ity & RCl cells, and a slight &m&tory effect on cell proliferation (pd.05 at 100nM). In RC2 cells, EGF had no effect or alk phos levels but significantly stimulated thymidine incorporation at all concentrations tested. RCl cells appeared more sensitive to vitamin A than RC2, with a 4.3 fold increase in thymidine incorporation at 1OOnM (p < 0.001) compared to only a 2.5 fold increase in RC2 cells (p < 0.001). Analysis of the Cytodex bead cultures showed that RC2 produced significantly greater mineralised material than RCI. These studies suggest that RCl and RC2 may be useful in vitro systems to examine the effect of growth factors on stromal cell differentiation. (1) ME Nuttall et al and (2) AJ Millest et al, Bone and Tooth Society, Sept 92
MRC Bone Research Luborutory, Nuftrerd Orthopaedic Centre, Headington, Oxford OX3 7LD and *British Bio-rechnotogy Ltd., Watlingfon Road, Cowley, Oxford OX4 5LY
P4. Modulation of the differentiation pattern of meeenchymal allr by bone morphogenetic proteins (BM.Ps) and parathyruid hormone 0 H Mayer, A Hollnagel, M Ahrens, T Ankenbauer, D Schroder, A Scutt, M Rohde, G Gross GBF, Moscheroder Weg I, D-3300 Braunschweig, Germany Osteoblast-chondroblast-adipocyte-differentiation potency of BMP’s and PTH was investigated. The cDNAs encoding hBM.P-2, 4, -5, -6, -7 in an eukaryotic expression vector were permanently transferred into the murine mesenchymal progenitor cell line C3HlOT1/2, known to differentiate into myotubes, adipocytes and chondrocytes upon the addition of azacytidine. Permanent transfection of genes encoding LBMP-2 to -7 induces differentiation. The osteogenic differentiation potential is substantiated by hisb3chemical and genetic analyses of marker
Hunt
Orrhopaedic
P6. Sex steroid* stimulate osteogenic differentiation by rat bone marrow cells A Scutt, P Scholz Schering AG, 1000 Berlin 65 Federal Republic of Germany
It is known that oestrogen deficiency during osteoporosis leads to an increase in osteoclast differentiation from bone marrow cells (BMSC) leading in turn to an increase in bone destruction. The purpose of this study was to see if sex steroids are also involved in the differentiation of osteoblasts from BMSC thus modulating bone formation. Rat BMSC were cultured at a density of 3.5 x 106 cells per cm2 in phenol red free DMEM
231 genes typical or specific for osteogenesis including the parathyroid hormone receptor alkaline phosphatase, osteopontin, osteonectin and osteocalcin. In addition to osteoblast-chondrocyte formation also differentiation into adipocytes is observed suggesting potential common progenitors for osteogenesis and adipogenesis. We investigated whether PTH in this system directs the differentiation into the osteogenic and/or other chondrogenic lineage. After expression of the rat PTH-receptor in BMP-~ansfected c3HlOT1/2 c&s adipogenesis is inhibited after addition of the liaand PTH (l-34). In addition surprisingly PTH (I-34) alone is s:fficient to induce nodul&ke structures in PTH-receptor transfected C3HlOT1/2 cells in the absence of BMP. Mineralisation was studied in bone marrow strornal cells. Element distribution and electron energy loss spectra images of phosphorus, calcium and oxygen show the localization of these elements within the same area indicating a chemical composition comparable to hydroxyapatite.
P5. In oitro properties of two immortalised rtromal cell lines derived from rabbit bone marrow E Brown, C Simpson, ME Nuttall*, B Ashton”, D Johnstone VIM Research Departmenf, Zeneca Pharmaceuticals, Mereside, Alderley Park Macclesfield Cheshire SK10 4TG; lSKB Phanmaceuticals, King of Prussia, PA. USA and **Department of
P3. Endothclial cells up-regulate alkaline phosphatarc expression by marrow stromal cells in co-culture RP Allsopp, AH Dnmunond*, LA N&ham*, JT Triffit
Rheumatology, Roberf Iones and Agnes Hospital, Oswestry, Shropshire SYlO 7AG
There is increasing interest in the role of endothelial cells in bone formatlon: the &se proximity of blood vessels to sites of active osteogenesis may reflect more than a requirement for a blood supply. Human marrow stromal (HM) cells were grown in co-culture for 5 days with bovine adrenal capillary endothelial (BACE) cells in DMIEM containing 10% foetal bovine serum, acidic fibroblast growth factor (long/ml) and heparin (15OOunits/ml). Histochemical and quantitative assays detected an increase in the HM-expression of alkaline phosphatase relative to that seen in the co-culture of HM and human skin cells or the culture of HM alone. If the BACE cells and HM cells were separated by a tissue culture Insert then no increase in alkaline phosphatase expression was seen. It is considered that HM cell populations, contain osteoprogenitors and alkaline phosphatase expression is an early marker of the osteogenic lineage. Our results suggest that endothelial cells are involved in specific interactions with HM cells causing differentiation towards the osteogenic phenotype. Further, this phenomenon requires cell-cell contact and is not solely mediated by the diffusion of soluble factors. Future work will investigate the mechanisms by which the two cell types interact.
Two cell clones, RCl and RC2, have been established by electroporation of rabbit bone marrow cells with a plasmid containing the temperature sensitive sv40 large T antigen and neomycin resistance genes (tsA58U19). RC2, but not RCI, has been shown to form bone in tivo; however, neither clone expressed significant levels of alkaline phosphatase (alk phos) in vitro even after dexamethasone or vitamin l& treatment (1). We have examined the effect of rhTGFg, rhEGF.and vitamin A on RCl and RC2 cell proliferation (measured bv thvmidine incorporation), and c;?llular differentiation (mea&red l& alk phos activity) in vitro, as well as examining their capacity to produce mineralised matrix when co-cultured with Cytodex beads (2). TGF8 (4-4OOpM) did not have any effect on RCl and RC2 aIk phos activity, bit inhibited cell proliferation in both clones at Sn&ml(~cO.OS>. EGF (0.1-1OOnM) caused an inhibition of alk ph& ac&ity & RCl cells, and a slight &m&tory effect on cell proliferation (pd.05 at 100nM). In RC2 cells, EGF had no effect or alk phos levels but significantly stimulated thymidine incorporation at all concentrations tested. RCl cells appeared more sensitive to vitamin A than RC2, with a 4.3 fold increase in thymidine incorporation at 1OOnM (p < 0.001) compared to only a 2.5 fold increase in RC2 cells (p < 0.001). Analysis of the Cytodex bead cultures showed that RC2 produced significantly greater mineralised material than RCI. These studies suggest that RCl and RC2 may be useful in vitro systems to examine the effect of growth factors on stromal cell differentiation. (1) ME Nuttall et al and (2) AJ Millest et al, Bone and Tooth Society, Sept 92
MRC Bone Research Luborutory, Nuftrerd Orthopaedic Centre, Headington, Oxford OX3 7LD and *British Bio-rechnotogy Ltd., Watlingfon Road, Cowley, Oxford OX4 5LY
P4. Modulation of the differentiation pattern of meeenchymal allr by bone morphogenetic proteins (BM.Ps) and parathyruid hormone 0 H Mayer, A Hollnagel, M Ahrens, T Ankenbauer, D Schroder, A Scutt, M Rohde, G Gross GBF, Moscheroder Weg I, D-3300 Braunschweig, Germany Osteoblast-chondroblast-adipocyte-differentiation potency of BMP’s and PTH was investigated. The cDNAs encoding hBM.P-2, 4, -5, -6, -7 in an eukaryotic expression vector were permanently transferred into the murine mesenchymal progenitor cell line C3HlOT1/2, known to differentiate into myotubes, adipocytes and chondrocytes upon the addition of azacytidine. Permanent transfection of genes encoding LBMP-2 to -7 induces differentiation. The osteogenic differentiation potential is substantiated by hisb3chemical and genetic analyses of marker
Hunt
Orrhopaedic
P6. Sex steroid* stimulate osteogenic differentiation by rat bone marrow cells A Scutt, P Scholz Schering AG, 1000 Berlin 65 Federal Republic of Germany
It is known that oestrogen deficiency during osteoporosis leads to an increase in osteoclast differentiation from bone marrow cells (BMSC) leading in turn to an increase in bone destruction. The purpose of this study was to see if sex steroids are also involved in the differentiation of osteoblasts from BMSC thus modulating bone formation. Rat BMSC were cultured at a density of 3.5 x 106 cells per cm2 in phenol red free DMEM