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Human primary osteocyte differentiation in a three-dimensional culture system
Abstracts / Bone 44 (2009) S253–S338
P125 Human primary osteocyte differentiation in a three-dimensional culture system F. Boukhechbaa,*, J. Michielsb, K. Ackermannc, D. Quinceya, J. Boulerd, W. Pyerinc, G.F. Carlea, N. Rocheta a GEPITOS, UMR 6235, Université Nice Sophia-Antipolis, CNRS b Service d'Anatomopathologie, Centre Hospitalier Universitaire de Nice, NICE, France c Biochemische Zellphysiologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany d LIOAD, UMR 791, Université de Nantes, INSERM, NANTES, France Introduction: Investigations on primary osteocytes, which compose over 90–95% of bone cells, embedded throughout the mineralized matrix, is a major challenge due to their difficult accessibility and the very rare models available in vitro. We engineered a threedimensional (3D) culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Materials and Methods: Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for four weeks in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Results: Histological analysis revealed, after only one week, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real time PCR expression of a set of bonerelated genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. Conclusions: This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a reliable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the crosstalk of osteocytes with tumor cells in bone metastasis since it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could be also a very efficient tool for drug testing and bone tissue engineering applications. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.551
P126 Cell-contacts are a prerequisite for basal and t3 regulated osteocalcin expression in mc3t3-e1 osteoblast like cells F. Vargaa,*, S. Spitzera, H. Karlicb, R. Thalera, K. Klaushofera a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidl, 4th Medical Department, Hanusch Hospital b Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria Osteocalcin (OCN), an osteoblast-specific protein, that was recently suggested to be involved in energy metabolism, is regulated by T3. It was demonstrated that the transcription of this gene
S301
depends on the transcription factor Runx2, which is indispensable for the differentiation of osteoblasts. Lymphoid enhancer protein-1 (Lef1) in cooperation with β-Catenin (βCat) inhibits the Runx2dependent transcription. βCat is a central protein in the Lrp5/Wntpathway that is important for bone metabolism. In addition this protein in cooperation with Cadherins is involved in formation of proper cell-contacts. During this process in endothelial cells βCat not only upregulates expression of E-Cadherin but also translocates from the nuclei to the cell contacts. MC3T3-E1 were seeded at a density of 2500 or 50,000/cm2 cultured over night and then treated with 100 nM T3 for 2 days or were left untreated for controls. Thereafter, RNA was isolated and was, after reverse transcription, subjected to quantitative PCR (QRTPCR). For immune-fluorescence cells were cultured as mentioned above, fixed with 4% paraformaldehyde and stained with monoclonal antibodies against β-Catenin or Pancadherin.mRNA expression studies revealed that in single cells OCN was nearly undetectable also by QRT-PCR and was stimulated by T3 weakly. In dense, confluent cultures basal expression increased up to 10 fold, regulation by T3 was found up to 30 fold. Both basal expression of osteocalcin as well stimulation by T3 strongly depended on the density of the cultures. In single cells βCat was localized mainly in the nuclei while after formation of cell contacts the protein was found on cell-contacts together with Cadherins. In dense cultures several genes important for the described regulatory events were upregulated: Runx2, βCat and OB-Cadherin and αT3-receptor, but not Lef1. This data indicate that the formation of cell contacts is important for basal as well as T3 regulated expression of OCN and other proteins of the osteoblastic phenotype. We speculate that in single cells basal expression is repressed by Lef1/βCat, while in confluent cultures the transcriptional suppressor function by βCat is switched of as this protein moves to the cell-contacts. T3 regulation will by enabled by upregulation of thyroid hormone receptor in dense cultures. In conclusion our data suggest that formation of proper cell contacts is a prerequisite for consequential mechanistic events in osteoblastic differentiation. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.552
P127 Long term oncostatin m treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells B. Brounaisa, E. Davida, C. Chipoyb, V. Tricheta, V. Ferréc, C. Charriera, L. Duplomba, M. Berreura, F. Rédinia, D. Heymanna, F. Blancharda,* a Inserm U957- EA3822, Faculté de Mécedine, Nantes, France b Molecular Oncology Group, McGill University, Montreal, Canada c Laboratoire d'Immunovirologie et Polymorphisme Génétique, Université de Nantes, Nantes, France Previous in vitro studies on primary osteoblastic and osteosarcoma cells (normal and transformed osteoblasts) have shown that Oncostatin M (OSM), a member of the interleukin-6 family, possesses both cytostatic and pro-apoptotic effects. On differentiation, this cytokine has complex and poorly understood activities, stimulating the first stages of osteogenesis but inhibiting osteoblast marker expression and bone formation at later stages. In rats, OSM also (i) reduces osteoblastic markers and induces trabecular bone loss and (ii) synergizes with other anti-cancer agents to induce apoptosis/necrosis in osteosarcoma cells. These effects were observed with short term OSM treatment for in vitro studies or with an adenoviral OSM gene transfer which induces only a transient and systemic overexpression of OSM in vivo. In this study, we analyze rat osteosarcoma cells transduced with lentiviral particles encoding OSM (lvOSM) to stably
P125 Human primary osteocyte differentiation in a three-dimensional culture system F. Boukhechbaa,*, J. Michielsb, K. Ackermannc, D. Quinceya, J. Boulerd, W. Pyerinc, G.F. Carlea, N. Rocheta a GEPITOS, UMR 6235, Université Nice Sophia-Antipolis, CNRS b Service d'Anatomopathologie, Centre Hospitalier Universitaire de Nice, NICE, France c Biochemische Zellphysiologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany d LIOAD, UMR 791, Université de Nantes, INSERM, NANTES, France Introduction: Investigations on primary osteocytes, which compose over 90–95% of bone cells, embedded throughout the mineralized matrix, is a major challenge due to their difficult accessibility and the very rare models available in vitro. We engineered a threedimensional (3D) culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Materials and Methods: Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for four weeks in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Results: Histological analysis revealed, after only one week, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real time PCR expression of a set of bonerelated genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. Conclusions: This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a reliable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the crosstalk of osteocytes with tumor cells in bone metastasis since it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could be also a very efficient tool for drug testing and bone tissue engineering applications. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.551
P126 Cell-contacts are a prerequisite for basal and t3 regulated osteocalcin expression in mc3t3-e1 osteoblast like cells F. Vargaa,*, S. Spitzera, H. Karlicb, R. Thalera, K. Klaushofera a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidl, 4th Medical Department, Hanusch Hospital b Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria Osteocalcin (OCN), an osteoblast-specific protein, that was recently suggested to be involved in energy metabolism, is regulated by T3. It was demonstrated that the transcription of this gene
S301
depends on the transcription factor Runx2, which is indispensable for the differentiation of osteoblasts. Lymphoid enhancer protein-1 (Lef1) in cooperation with β-Catenin (βCat) inhibits the Runx2dependent transcription. βCat is a central protein in the Lrp5/Wntpathway that is important for bone metabolism. In addition this protein in cooperation with Cadherins is involved in formation of proper cell-contacts. During this process in endothelial cells βCat not only upregulates expression of E-Cadherin but also translocates from the nuclei to the cell contacts. MC3T3-E1 were seeded at a density of 2500 or 50,000/cm2 cultured over night and then treated with 100 nM T3 for 2 days or were left untreated for controls. Thereafter, RNA was isolated and was, after reverse transcription, subjected to quantitative PCR (QRTPCR). For immune-fluorescence cells were cultured as mentioned above, fixed with 4% paraformaldehyde and stained with monoclonal antibodies against β-Catenin or Pancadherin.mRNA expression studies revealed that in single cells OCN was nearly undetectable also by QRT-PCR and was stimulated by T3 weakly. In dense, confluent cultures basal expression increased up to 10 fold, regulation by T3 was found up to 30 fold. Both basal expression of osteocalcin as well stimulation by T3 strongly depended on the density of the cultures. In single cells βCat was localized mainly in the nuclei while after formation of cell contacts the protein was found on cell-contacts together with Cadherins. In dense cultures several genes important for the described regulatory events were upregulated: Runx2, βCat and OB-Cadherin and αT3-receptor, but not Lef1. This data indicate that the formation of cell contacts is important for basal as well as T3 regulated expression of OCN and other proteins of the osteoblastic phenotype. We speculate that in single cells basal expression is repressed by Lef1/βCat, while in confluent cultures the transcriptional suppressor function by βCat is switched of as this protein moves to the cell-contacts. T3 regulation will by enabled by upregulation of thyroid hormone receptor in dense cultures. In conclusion our data suggest that formation of proper cell contacts is a prerequisite for consequential mechanistic events in osteoblastic differentiation. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.552
P127 Long term oncostatin m treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells B. Brounaisa, E. Davida, C. Chipoyb, V. Tricheta, V. Ferréc, C. Charriera, L. Duplomba, M. Berreura, F. Rédinia, D. Heymanna, F. Blancharda,* a Inserm U957- EA3822, Faculté de Mécedine, Nantes, France b Molecular Oncology Group, McGill University, Montreal, Canada c Laboratoire d'Immunovirologie et Polymorphisme Génétique, Université de Nantes, Nantes, France Previous in vitro studies on primary osteoblastic and osteosarcoma cells (normal and transformed osteoblasts) have shown that Oncostatin M (OSM), a member of the interleukin-6 family, possesses both cytostatic and pro-apoptotic effects. On differentiation, this cytokine has complex and poorly understood activities, stimulating the first stages of osteogenesis but inhibiting osteoblast marker expression and bone formation at later stages. In rats, OSM also (i) reduces osteoblastic markers and induces trabecular bone loss and (ii) synergizes with other anti-cancer agents to induce apoptosis/necrosis in osteosarcoma cells. These effects were observed with short term OSM treatment for in vitro studies or with an adenoviral OSM gene transfer which induces only a transient and systemic overexpression of OSM in vivo. In this study, we analyze rat osteosarcoma cells transduced with lentiviral particles encoding OSM (lvOSM) to stably