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Hypothermia stimulates osteoclastogenesis but inhibits osteoblast differentiation and bone formation
Abstracts / Bone 44 (2009) S253–S338
in the bone marrow (Mesenchymal Stem Cells, MSC), we focused on regulatory events occur during lineage decision/commitment. This could further explain whether an increased number of bone marrow adipocytes is caused by a defect in lineage commitment during MSC differentiation. To elucidate this question we have differentiated bone marrow derived human MSCs (CD105+, CD166+, CD29+,CD44+ and CD14-, CD34-, CD45-) into osteoblasts and adipocytes. Cells were harvested for RNA and biochemical analyses at 0 h, 0.5 h, 1 h, 2 h, 3 h, 6 h, 12 h, 1 day, 2d, 3d, 4d, 5d, 7d, 10d, 13d, 17d, 21d, 25d after induction of differentiation into adipocyte or osteoblast. RNA from these cultures was amplified, labeled and hybridized on a Illumina HumanHT-12 expression array containing around 48000 different probes. After scanning, data was extracted and normalized. Global analysis illustrates very dynamic transcriptional changes during differentiation into adipocytes and osteoblast. Most changes occur within the first 2 days of both cultures. After 3 h about 400 genes were significantly (p < 0.0001) different expressed in the osteogenic and adipogenic lineage which increased to 2000 transcripts after 2 days. These contained interesting clusters of transcription factors/regulators and other regulatory proteins such as kinases/phosphatases. In osteoblast differentiation we identified a stable gene expression period of 2 weeks which was followed by a very dynamic expression period during mineralization. During this stage, cells produce extra-cellular matrix proteins and getting prepared for mineralization. Comparative profile analyses showed limited lineage specificity of known markers (e.g. osteoblast ALPL and RUNX2) but also delivered novel and more specific marker genes for early detection of lineage commitment. Taken together the transcriptional changes illustrate specific expression pattern for differentiation indicating early lineage commitment. More in depth analyses will follow and will identify and functional characterize novel regulatory mechanisms of hMSC differentiation and our understanding in lineage decision/commitment. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.561
P136 Hypothermia stimulates osteoclastogenesis but inhibits osteoblast differentiation and bone formation J.J. Patela,*, I.R. Orrissa, M.L. Keya, S.E.B. Taylora, K. Karnikb, T.R. Arnetta a Department of Cell and Developmental Biology, University College London, London b Unilever Discover, Unilever PLC, Sharnbrook, UK Core body temperature declines with aging due to a reduction in heat production and peripheral vasoconstriction. Core body temperatures < 35.5 °C are common in the elderly population. The effects of chronic hypothermia on bone cell function and skeletal homeostasis have been little studied. The aim of this study was to investigate the effects of hypothermia (34 °C) and mild hypothermia (35.5 °C) on osteoblasts and osteoclasts. Primary osteoblast cultures were derived from neonatal rat calvaria by trypsin/ collagenase digestion and cultured for 14–16 days in medium supplemented with ascorbate, beta-glycerophosphate and dexamethasone. Osteoclast-forming mononuclear cells were isolated from long bone marrow of 6–8 week old mice and cultured on ivory discs with MCSF and RANKL for 8 days, with acidification to pH 6.9 for the final 2 days to activate resorption. We found that cell number was initially reduced by 20–30% in osteoblasts cultured at either 34 °C or 35.5 °C but recovered progressively, reaching control
S305
levels after 14 days. The expression of osteocalcin, alkaline phosphatase, type I collagen and osteopontin mRNAs in osteoblasts cultured at either 34 °C or 35.5 °C showed a similar pattern of initial downregulation followed by recovery; the same trend was observed for soluble collagen and alkaline phosphatase activity. However, formation of 'trabecular' bone nodules, assessed morphometrically following alizarin red staining, decreased by 95% and 75% at 34 °C and 35.5 °C respectively, compared to 37 °C. In contrast, we found that osteoclast cell number and resorption pit formation increased 1.5 to 2-fold at 34 °C or 35.5 °C, compared to 37 °C. Thus, both hypothermia and mild hypothermia exert reciprocal effects on bone cell function by retarding osteoblast proliferation and differentiation, leading to reduced bone formation, whilst causing a modest stimulation of osteoclastogenesis and resorption. These results suggest that hypothermia in the elderly could help shift the bone remodelling balance in the negative direction, contributing to the pathogenesis of osteoporosis. Conflict of interest: K. Karnik, Unilever Discover, Grant Research Support. doi:10.1016/j.bone.2009.03.562
P137 Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin J. Kühnischa,*, M. Kolanczykb, N. Kosslerb, S. Stumppa, M. Osswaldb, B. Thurischb, U. Kornaka, S. Mundlosa a Institut für Medizinische Genetik, Charite b FG Development and Disease, Max-Planck Institute for Molecular Genetics, Berlin, Germany Neurofibromatosis type 1 (NF1) is characterised by the appearance of skin freckling, café-au-lait spots, and Schwann cell tumours (neurofibroma). However, about 50% of the NF1 patients are also affected by skeletal abnormalities such as scoliosis, osteoporosis, and tibia bowing. It was shown that loss of neurofibromin, the protein encoded by the NF1 gene, causes tibia bowing due to reduced bone material quality. Tibia bowing and local bone weakening may finally result in fractures and pseudarthrotic lesions which are very resistant to treatment. As Neurofibromin is a negative regulator of Ras activity and statins act, by down-regulating Ras activity, in an osteoanabolic manner lovastatin might improve fracture healing after loss of NF1. In order to investigate a) the role of Nf1 in fracture healing and b) the therapeutic potential of lovastatin we established a cortical defect model in a mouse mutant with limb-specific ablation of neurofibromin (Nf1Prx1). Bone injury restoration was severely delayed in Nf1Prx1 drilling defects due to excessive formation as well as persistence of fibro-cartilaginous tissue. Moreover, reduced extracellular matrix mineralisation was caused by defective osteoblast differentiation due to diminished Runx2 expression. Systemic treatment with lovastatin improved cortical and medullar bone formation in Nf1Prx1 mice, increased Runx2 expression and lowered mitogen activated protein kinase activity, resulting in enhanced osteoblast differentiation. In summary, this pre-clinical study establishes a positive influence of lovastatin on bone fracture healing in a neurofibromin knock-out mouse model. Therefore, lovastatin treatment provides a potential pharmaceutical approach to improve fracture healing also in NF1 patients. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.563
in the bone marrow (Mesenchymal Stem Cells, MSC), we focused on regulatory events occur during lineage decision/commitment. This could further explain whether an increased number of bone marrow adipocytes is caused by a defect in lineage commitment during MSC differentiation. To elucidate this question we have differentiated bone marrow derived human MSCs (CD105+, CD166+, CD29+,CD44+ and CD14-, CD34-, CD45-) into osteoblasts and adipocytes. Cells were harvested for RNA and biochemical analyses at 0 h, 0.5 h, 1 h, 2 h, 3 h, 6 h, 12 h, 1 day, 2d, 3d, 4d, 5d, 7d, 10d, 13d, 17d, 21d, 25d after induction of differentiation into adipocyte or osteoblast. RNA from these cultures was amplified, labeled and hybridized on a Illumina HumanHT-12 expression array containing around 48000 different probes. After scanning, data was extracted and normalized. Global analysis illustrates very dynamic transcriptional changes during differentiation into adipocytes and osteoblast. Most changes occur within the first 2 days of both cultures. After 3 h about 400 genes were significantly (p < 0.0001) different expressed in the osteogenic and adipogenic lineage which increased to 2000 transcripts after 2 days. These contained interesting clusters of transcription factors/regulators and other regulatory proteins such as kinases/phosphatases. In osteoblast differentiation we identified a stable gene expression period of 2 weeks which was followed by a very dynamic expression period during mineralization. During this stage, cells produce extra-cellular matrix proteins and getting prepared for mineralization. Comparative profile analyses showed limited lineage specificity of known markers (e.g. osteoblast ALPL and RUNX2) but also delivered novel and more specific marker genes for early detection of lineage commitment. Taken together the transcriptional changes illustrate specific expression pattern for differentiation indicating early lineage commitment. More in depth analyses will follow and will identify and functional characterize novel regulatory mechanisms of hMSC differentiation and our understanding in lineage decision/commitment. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.561
P136 Hypothermia stimulates osteoclastogenesis but inhibits osteoblast differentiation and bone formation J.J. Patela,*, I.R. Orrissa, M.L. Keya, S.E.B. Taylora, K. Karnikb, T.R. Arnetta a Department of Cell and Developmental Biology, University College London, London b Unilever Discover, Unilever PLC, Sharnbrook, UK Core body temperature declines with aging due to a reduction in heat production and peripheral vasoconstriction. Core body temperatures < 35.5 °C are common in the elderly population. The effects of chronic hypothermia on bone cell function and skeletal homeostasis have been little studied. The aim of this study was to investigate the effects of hypothermia (34 °C) and mild hypothermia (35.5 °C) on osteoblasts and osteoclasts. Primary osteoblast cultures were derived from neonatal rat calvaria by trypsin/ collagenase digestion and cultured for 14–16 days in medium supplemented with ascorbate, beta-glycerophosphate and dexamethasone. Osteoclast-forming mononuclear cells were isolated from long bone marrow of 6–8 week old mice and cultured on ivory discs with MCSF and RANKL for 8 days, with acidification to pH 6.9 for the final 2 days to activate resorption. We found that cell number was initially reduced by 20–30% in osteoblasts cultured at either 34 °C or 35.5 °C but recovered progressively, reaching control
S305
levels after 14 days. The expression of osteocalcin, alkaline phosphatase, type I collagen and osteopontin mRNAs in osteoblasts cultured at either 34 °C or 35.5 °C showed a similar pattern of initial downregulation followed by recovery; the same trend was observed for soluble collagen and alkaline phosphatase activity. However, formation of 'trabecular' bone nodules, assessed morphometrically following alizarin red staining, decreased by 95% and 75% at 34 °C and 35.5 °C respectively, compared to 37 °C. In contrast, we found that osteoclast cell number and resorption pit formation increased 1.5 to 2-fold at 34 °C or 35.5 °C, compared to 37 °C. Thus, both hypothermia and mild hypothermia exert reciprocal effects on bone cell function by retarding osteoblast proliferation and differentiation, leading to reduced bone formation, whilst causing a modest stimulation of osteoclastogenesis and resorption. These results suggest that hypothermia in the elderly could help shift the bone remodelling balance in the negative direction, contributing to the pathogenesis of osteoporosis. Conflict of interest: K. Karnik, Unilever Discover, Grant Research Support. doi:10.1016/j.bone.2009.03.562
P137 Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin J. Kühnischa,*, M. Kolanczykb, N. Kosslerb, S. Stumppa, M. Osswaldb, B. Thurischb, U. Kornaka, S. Mundlosa a Institut für Medizinische Genetik, Charite b FG Development and Disease, Max-Planck Institute for Molecular Genetics, Berlin, Germany Neurofibromatosis type 1 (NF1) is characterised by the appearance of skin freckling, café-au-lait spots, and Schwann cell tumours (neurofibroma). However, about 50% of the NF1 patients are also affected by skeletal abnormalities such as scoliosis, osteoporosis, and tibia bowing. It was shown that loss of neurofibromin, the protein encoded by the NF1 gene, causes tibia bowing due to reduced bone material quality. Tibia bowing and local bone weakening may finally result in fractures and pseudarthrotic lesions which are very resistant to treatment. As Neurofibromin is a negative regulator of Ras activity and statins act, by down-regulating Ras activity, in an osteoanabolic manner lovastatin might improve fracture healing after loss of NF1. In order to investigate a) the role of Nf1 in fracture healing and b) the therapeutic potential of lovastatin we established a cortical defect model in a mouse mutant with limb-specific ablation of neurofibromin (Nf1Prx1). Bone injury restoration was severely delayed in Nf1Prx1 drilling defects due to excessive formation as well as persistence of fibro-cartilaginous tissue. Moreover, reduced extracellular matrix mineralisation was caused by defective osteoblast differentiation due to diminished Runx2 expression. Systemic treatment with lovastatin improved cortical and medullar bone formation in Nf1Prx1 mice, increased Runx2 expression and lowered mitogen activated protein kinase activity, resulting in enhanced osteoblast differentiation. In summary, this pre-clinical study establishes a positive influence of lovastatin on bone fracture healing in a neurofibromin knock-out mouse model. Therefore, lovastatin treatment provides a potential pharmaceutical approach to improve fracture healing also in NF1 patients. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.563