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P13. Immunocytochemistry of cultured human trabecular bone stromal cells
120
Abstracts
angiogenic process. The amounts needed for enzyme activation do indicate its genuine importance in the connective tissue degradation process. We are grateful to T. Cawston, G. Murphy & H. Nagase for TIMP-2, Pro MMP-2 and Pro MMI’-3 respectively.
P14. Gap junctions between osteoblast-like cells in vitro: A model to study cell-cell-communication K Schirrmacher, E Winterhager*, 0 Traub”, F Brummer+, D Jones++ and D Bingmann Institute fur Physiologic und fur ‘Anatomic, Hufeland str. 55, D4300 Essen; “Institut +Biologisches Institut,
Pl2. Dcxtran sulphate promotes stromrl cell aggregation initiates type II collagen production BMThomson, V Dean, B Noble and N Loveridge Rowett Research Institute, Aberdeen
and
Porcine bone-marrow stromal cells form cartilage in diffusion chambers in uiuo, but do not express type II collagen in monolayer culture. Since glycoconjugates may regulate chondrogenesis, we studied the effect of a glycosaminoglycan analogue, dextran sulphate (DxS), on stromal cell differentiation. DxS induced confluent porcine bone-marrow stromal-cell monolayers to retract into tightly packed circular aggregates (approx 80-200 cells; 252 f 21 mm diameter; 96.5 f 5.5 clusters/ cm?). Retraction began within 6h and was complete after 3-5 days. Dye exclusion indicated continued cell viability and DNA content was unchanged over 24h Dextran 500, chondroitin sulphate, dermatan sulphate and sodium sulphate were without effect. Dexamethasone, ascorbate and B-glycerophosphate (DAB), which induce mineralised nodule formation over lo-12 days, produced no morphological change over 6 days, but increased proliferation and aggregation in DxS treated cultures. Immunocytochemistry revealed type I but not type II collagen production by monolayer cultures. DAb increased type I collagen deposition but not type Il production over 6 days. In contrast, cell aggregates formed in the presence of DxS or DxS/DAb expressed type II collagen in their central regions. Type I was immunolocalised to the aggregate’s peripheries but was conspicuously absent from their centres. This data suggests chondrocytic differentiation occurs within stromal cell aggregates. We believe this is the first indication chondrogenesis from mammalian stromal cells in vitro.
P13. Immunocytochemistry of cultured human trabecular bone stmmal cells JMW Quinn, S Graves’, MJ Francis* and NA Athanasou* University of Oxford, Nuffield Department of Pathology and Bacteriology, john Radcliffe Hospital, Oxford OX3 9DU and ‘Department of Pathology, Nuffield Orthopaedic Centre, Oxford Primary cultures of human trabecular bone stromal cells (TBSCs) provide a useful model of matrix mineralisation by human osteoblasts. We analysed immunocytochemically the antigenic phenotype of TBSCs to determine if contaminating haemopoietic and endothelial cells are present in such cultures and to determine the range of cell adhesion molecules (CAMS) present on such cells under different mineralizing conditions. Cells were derived from specimens of human trabecular bone, with skin fibroblasts from the same patients as control. Both spindle shape TBSCs and fibroblasts expressed &I integrins VLA2, VLA-4 (but not VLA-1, VLA5, VLA-6, or b-2 or 83 integrins), CD44 (H-CAM), CD13 and vimentin. CD4 (LCA) and CD14 were not expressed by spindle-shape TBSCs but scanty (< 0.1%) positive round myeloid cells were found even after passage. Unlike fibroblasts, a minor fraction of TBSCs (-z 5%) expressed HLA-DR. Endothelial markers were not expressed. Addition of ascorbate-2-phosphate did not alter the antigenic phenotype. This indicates that this methodology produces relatively pure populations of osteoblast-like human TBSCs. TBSCs an fibroblasts showed a restricted expression of CAMs including receptors for collagen and fibronectin. The minor fraction of HLA-DR positive TBSCs may reflect culture conditions or indicate that a distinct type of bone stromal cell exists.
from the Joint Meeting, September 1992
++lnstitut
fur Genetik, Romerstr. 264, D-5300 Bonn; Pfaffn-waldring 57, D-7000 Stuttgart 80;
fur Zell-biologi?,
Domagkstr.
3, D-4400 Munster
It is well known that bone cells in tivo are interconnected by gap junctions which may mediate the transmission of hormonal or bioelectric signals. Such gap junctions exist also in vitro. The aim was to analyse properties of these gap junctions in cultures of osteoblast-like cells COB) derived from calvarial fragments of new-born rats and of guinea pigs. In vitro migration of 08 from calvarial fragments was observed 2-4 weeks after explantation. Immunocytochemical investigations and Western blotting revealed connexin43 (cx43 as the major gap junction protein which has also be found between cardiac or astrocytic cells. In the present studies the existence of functional connections between OB in primary culture was shown by dye coupling with the fluorescent tracer Lucifer yellow (n=SO) and electric coupling (n=71). Electric coupling was rather stable during changes of voltage, rise in the extracellular Ca 2+ concentration, acidification and increase of the intracellular CAMP level. Gap junction coupling was blocked by octanol (lo-4 mol/l). Calcitonin (20 mLJ/ml) was found to reduce or improve electric coupling between OB. Thus, regulatory properties of cx43 gap junction channels in OB differ from those of cardiac and astrocytic cx43 gap junctions, indicating that the properties of gap junctions do not only depend on the type of connexin. Supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG Bi 278/6-l). p15. Expression of tenascin by osteoblaatic cells in oitro and in nioo EJ Ma&e and RP Tucker’ Rheumatology Research Unit, Addenbrooke’s Hospital, Cambridge and ‘Department of Neurobiology Anatomy, Bowman Gray School of Medicine, Winston-Salem, NC, USA The expression of tenascin by human and rat osteoblast-like cell lines and primary osteoblast-enriched cultures from chick embryo calvarial bones has been investigated. Cultured osteoblasts secreted tenascin into the medium and deposited it in their extracellular matrix. Osteoblast-conditioned medium contained almost exclusively the largest tenascin subunit, whereas medium from fibroblasts cultured from periostea of chick calvariae contained approximately equal amounts of all three subunits. Thus osteoblastic differentiation is associated with a change in the expression pattern of tenascin splice variants. In primary chick osteoblast-enriched cultures, extracellular matrix tenascin staining was only found in areas of alkaline phosphatase staining. Results of immunocytochemical studies suggested that incorporation of tenascin into fibrils in the extracellular matrix of cultured osteoblast-like cells depends on the presence of pre-existing fibronectin fibrils. In embryonic chicken bones, in situ hybridization studies demonstrated that osteoblasts also express tenascin in viuo. Tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable, indicating that turnover of tenascin occurs more rapidly in bone than in cartilage.
P16. Bigger is bettel: is this true for osteoclasts? A Boyde, SJ Jones, K Piper and S Komiya’ Dept of Anromy and Developmental Biology, University London, London WC/E 6BT and ‘Lasert Corporation, W12 9RT
College London
Abstracts
angiogenic process. The amounts needed for enzyme activation do indicate its genuine importance in the connective tissue degradation process. We are grateful to T. Cawston, G. Murphy & H. Nagase for TIMP-2, Pro MMP-2 and Pro MMI’-3 respectively.
P14. Gap junctions between osteoblast-like cells in vitro: A model to study cell-cell-communication K Schirrmacher, E Winterhager*, 0 Traub”, F Brummer+, D Jones++ and D Bingmann Institute fur Physiologic und fur ‘Anatomic, Hufeland str. 55, D4300 Essen; “Institut +Biologisches Institut,
Pl2. Dcxtran sulphate promotes stromrl cell aggregation initiates type II collagen production BMThomson, V Dean, B Noble and N Loveridge Rowett Research Institute, Aberdeen
and
Porcine bone-marrow stromal cells form cartilage in diffusion chambers in uiuo, but do not express type II collagen in monolayer culture. Since glycoconjugates may regulate chondrogenesis, we studied the effect of a glycosaminoglycan analogue, dextran sulphate (DxS), on stromal cell differentiation. DxS induced confluent porcine bone-marrow stromal-cell monolayers to retract into tightly packed circular aggregates (approx 80-200 cells; 252 f 21 mm diameter; 96.5 f 5.5 clusters/ cm?). Retraction began within 6h and was complete after 3-5 days. Dye exclusion indicated continued cell viability and DNA content was unchanged over 24h Dextran 500, chondroitin sulphate, dermatan sulphate and sodium sulphate were without effect. Dexamethasone, ascorbate and B-glycerophosphate (DAB), which induce mineralised nodule formation over lo-12 days, produced no morphological change over 6 days, but increased proliferation and aggregation in DxS treated cultures. Immunocytochemistry revealed type I but not type II collagen production by monolayer cultures. DAb increased type I collagen deposition but not type Il production over 6 days. In contrast, cell aggregates formed in the presence of DxS or DxS/DAb expressed type II collagen in their central regions. Type I was immunolocalised to the aggregate’s peripheries but was conspicuously absent from their centres. This data suggests chondrocytic differentiation occurs within stromal cell aggregates. We believe this is the first indication chondrogenesis from mammalian stromal cells in vitro.
P13. Immunocytochemistry of cultured human trabecular bone stmmal cells JMW Quinn, S Graves’, MJ Francis* and NA Athanasou* University of Oxford, Nuffield Department of Pathology and Bacteriology, john Radcliffe Hospital, Oxford OX3 9DU and ‘Department of Pathology, Nuffield Orthopaedic Centre, Oxford Primary cultures of human trabecular bone stromal cells (TBSCs) provide a useful model of matrix mineralisation by human osteoblasts. We analysed immunocytochemically the antigenic phenotype of TBSCs to determine if contaminating haemopoietic and endothelial cells are present in such cultures and to determine the range of cell adhesion molecules (CAMS) present on such cells under different mineralizing conditions. Cells were derived from specimens of human trabecular bone, with skin fibroblasts from the same patients as control. Both spindle shape TBSCs and fibroblasts expressed &I integrins VLA2, VLA-4 (but not VLA-1, VLA5, VLA-6, or b-2 or 83 integrins), CD44 (H-CAM), CD13 and vimentin. CD4 (LCA) and CD14 were not expressed by spindle-shape TBSCs but scanty (< 0.1%) positive round myeloid cells were found even after passage. Unlike fibroblasts, a minor fraction of TBSCs (-z 5%) expressed HLA-DR. Endothelial markers were not expressed. Addition of ascorbate-2-phosphate did not alter the antigenic phenotype. This indicates that this methodology produces relatively pure populations of osteoblast-like human TBSCs. TBSCs an fibroblasts showed a restricted expression of CAMs including receptors for collagen and fibronectin. The minor fraction of HLA-DR positive TBSCs may reflect culture conditions or indicate that a distinct type of bone stromal cell exists.
from the Joint Meeting, September 1992
++lnstitut
fur Genetik, Romerstr. 264, D-5300 Bonn; Pfaffn-waldring 57, D-7000 Stuttgart 80;
fur Zell-biologi?,
Domagkstr.
3, D-4400 Munster
It is well known that bone cells in tivo are interconnected by gap junctions which may mediate the transmission of hormonal or bioelectric signals. Such gap junctions exist also in vitro. The aim was to analyse properties of these gap junctions in cultures of osteoblast-like cells COB) derived from calvarial fragments of new-born rats and of guinea pigs. In vitro migration of 08 from calvarial fragments was observed 2-4 weeks after explantation. Immunocytochemical investigations and Western blotting revealed connexin43 (cx43 as the major gap junction protein which has also be found between cardiac or astrocytic cells. In the present studies the existence of functional connections between OB in primary culture was shown by dye coupling with the fluorescent tracer Lucifer yellow (n=SO) and electric coupling (n=71). Electric coupling was rather stable during changes of voltage, rise in the extracellular Ca 2+ concentration, acidification and increase of the intracellular CAMP level. Gap junction coupling was blocked by octanol (lo-4 mol/l). Calcitonin (20 mLJ/ml) was found to reduce or improve electric coupling between OB. Thus, regulatory properties of cx43 gap junction channels in OB differ from those of cardiac and astrocytic cx43 gap junctions, indicating that the properties of gap junctions do not only depend on the type of connexin. Supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG Bi 278/6-l). p15. Expression of tenascin by osteoblaatic cells in oitro and in nioo EJ Ma&e and RP Tucker’ Rheumatology Research Unit, Addenbrooke’s Hospital, Cambridge and ‘Department of Neurobiology Anatomy, Bowman Gray School of Medicine, Winston-Salem, NC, USA The expression of tenascin by human and rat osteoblast-like cell lines and primary osteoblast-enriched cultures from chick embryo calvarial bones has been investigated. Cultured osteoblasts secreted tenascin into the medium and deposited it in their extracellular matrix. Osteoblast-conditioned medium contained almost exclusively the largest tenascin subunit, whereas medium from fibroblasts cultured from periostea of chick calvariae contained approximately equal amounts of all three subunits. Thus osteoblastic differentiation is associated with a change in the expression pattern of tenascin splice variants. In primary chick osteoblast-enriched cultures, extracellular matrix tenascin staining was only found in areas of alkaline phosphatase staining. Results of immunocytochemical studies suggested that incorporation of tenascin into fibrils in the extracellular matrix of cultured osteoblast-like cells depends on the presence of pre-existing fibronectin fibrils. In embryonic chicken bones, in situ hybridization studies demonstrated that osteoblasts also express tenascin in viuo. Tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable, indicating that turnover of tenascin occurs more rapidly in bone than in cartilage.
P16. Bigger is bettel: is this true for osteoclasts? A Boyde, SJ Jones, K Piper and S Komiya’ Dept of Anromy and Developmental Biology, University London, London WC/E 6BT and ‘Lasert Corporation, W12 9RT
College London