- Email: [email protected]
Strontium ranelate fully prevents alteration of bone mechanical properties in response to cyclical loading
S66
Abstracts
a
Department of Exercise Sciences/Multidisciplinary Osteoporosis Research Program, University of Toronto/Women's College Hospital, Toronto, Canada b Quantum Medical Metrics LLC, USA c Department of Electrical and Computer Engineering, Johns Hopkins University, Baltimore, USA d Department of Medicine/Multidisciplinary Osteoporosis Research Program, University of Toronto/Women's College Hospital, Toronto, Canada e Department of Medicine, St. Joseph's Healthcare/McMaster University, Hamilton, Canada f Centre de Recherche du CHUQ, Laval University, Quebec City, Canada Abstract: The function of skeletal adaptation to mechanical load (modeling) is to adjust the amount and distribution of bone tissue (geometry) and perhaps material elastic properties, so that strains experienced within the bone are kept within certain physiological limits. Genetic, environmental or hormonal factors may cause heterogeneity in load response (mechanosensitivity), such that equivalent loads would generate higher or lower strains. Individuals who have reduced mechanosensitivity should require higher strains to generate an adaptive response, consequently have weaker bones and fracture earlier and more frequently throughout their life. The purpose of the current study was to determine if stresses (proportional to strains) at the femoral neck under equivalent physiologic loads were higher in women whose first fracture occurred early in life, than in women whose first fracture occurred late in life. We studied 3197 women between the ages of 25 and 92 who participated in the Canadian Multicentre Osteoporosis Study (CaMos) and who had available Hip Structure Analysis (HSA) data from baseline dual energy X-ray absorptiometry (DXA) scans. Women were categorized into 3 groups based on their age at first fracture (b 50 years, between 50–64 years and ≥65 years). We computed stress (megapascals = MPa) at the infero-medial margin of the femoral neck in a one-legged stance mode using a 2-D engineering beam analysis incorporating dimensions and geometry from DXA scans using the HSA method. We also assessed geometry parameters from the HSA analysis including: narrow neck (NN) bone mineral density (BMD), NN cross-sectional area (CSA) and NN section modulus (Z; an index of bending strength). We used general linear models (SAS 9.1) to determine associations between stance stress, geometry parameters and age at first fracture based on fracture groupings. We found that women whose first fracture was early in life (b50 years) had higher stress (10.35 ± 2.19 vs. 10.00 ± 1.99 MPa; p = 0.0468), lower NN BMD (0.757 ± 0.15 vs. 0.784 ± 0.16 g/cm2; p = 0.0306), lower NN CSA (1.895 ± 0.38 vs. 1.997 ± 0.42 cm2; p = 0.0013) and lower NN Z (0.912 ± 0.22 vs. 0.966 ± 0.25 cm3; p = 0.0024) than women whose first fracture was late in life (≥ 65 years). These findings provide evidence of heterogeneity in load response and suggest an important role for modeling in the pathogenesis of bone fragility in women. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: C. Hamilton: none declared, T. Beck Shareholder of Hologic Inc., A. Khaled: none declared, S. Jamal Advisory Board Membership of Novartis, Amgen, Warner-Chilcott, Consulting fees from Novartis, Warner-Chilcott, Genzyme, Shire, Speaker Fees from Novartis, Amgen, Warner-Chilcott, Genzyme, Shire, J. Adachi Grant / Research Support from Amgen, Bristol-Myers Squibb, Eli Lilly, Merck, Novartis, Pfizer, Procter & Gamble, Sanofi Aventis, Roche Warner-Chilcott, Consulting fees from Amgen, Eli Lilly, Glaxo Smith Kline, Merck, Novartis, Pfizer, Procter & Gamble, Roche, Sanofi Aventis, Warner-Chilcott, Speaker Fees from Amgen, Eli Lilly, GlaxoSmithKline, Merck, Novartis, Pfizer, Procter & Gamble, Roche, Sanofi Aventis, Warner-Chilcott, J. Brown Grant / Research Support from Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, Sanofi-Aventis, Servier, Warner-Chilcott, Speakers Bureau with Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, SanofiAventis, Servier, Warner-Chilcott, Consulting fees from Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, Sanofi-Aventis, Servier, WarnerChilcott, Speaker Fees from Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, Sanofi-Aventis, Servier, Warner-Chilcott, K. Davison Advisory Board Membership of Amgen, Servier, Novartis, Consulting fees from Merck, Amgen, Novartis, Servier, Speaker Fees from Merck, Warner-Chilcott, Amgen, Novartis, Servier.
doi:10.1016/j.bone.2012.02.183
NIOP04 / PP438 Active vitamin D3 administration improves locomotive ability of mice with rotarod training S. Sakai⁎, M. Suzuki, K. Tanaka, M. Mihara, K. Endo Product Research Department, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan Abstract: Active vitamin D3 improves bone mineral density and bone strength. Moreover, some studies have found that vitamin D3 decreases the risk of falls. However, we have very little knowledge about the relationship between active vitamin D3 and peripheral nerve function, which is known to decline with age. In this study, we used vitamin D receptor knockout (VDR KO) mice to investigate the effect of VDR
signaling on motor function. In addition, we evaluated the effect of a new antiosteoporotic medicine, eldecalcitol (ED-71), an active vitamin D3 analog, on enhancing locomotive ability in mice with rotarod training. Wild type (WT) and VDR KO mice were trained on a rotarod 5 days a week (15 rpm, 5 min, 3 times/day) for 4 weeks, and locomotive performance was then measured by an accelerating rotarod test. Four-week training led to an increased duration on the rotarod in the WT mice but not in the VDR KO mice, suggesting that VDR signaling is essential for the improvement of motor function. Histological observations of sciatic nerve cross sections revealed that the axon diameter was smaller in VDR KO mice than in WT mice. Additionally, the distribution of acetylcholine receptor on the extensor digitorum longus muscle was not as broad and the pattern of distribution was simpler in the VDR KO mice. Furthermore, active vitamin D3 dose-dependently upregulated mRNA expression of insulin-like growth factor-I (IGF-I) and myelin basic protein (MBP) in Schwann cells isolated from neonatal rats. We assessed the effect of ED-71 administration on locomotive ability of mice during rotarod training. Nine-week-old male C57BL mice either receiving or not receiving orally administered ED-71 were trained on a rotarod following the same training regimen as above for 2 weeks. The locomotive ability of trained mice receiving ED-71 was superior than that of those not receiving ED-71 treatment. In conclusion, VDR signaling plays a critical role in upregulating motor function in the rotarod training model. Stimulation with active vitamin D3 increased mRNA expression of IGF-I and MBP in Schwann cells in vitro. These results suggest that the improved motor function observed following administration of ED-71 may be via the induction of IGF-I and MBP from peripheral Schwann cells. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: S. Sakai Employee of Chugai Pharmaceutical Co., Ltd., M. Suzuki Employee of Chugai Pharmaceutical Co., Ltd., K. Tanaka Employee of Chugai Pharmaceutical Co., Ltd., M. Mihara Employee of Chugai Pharmaceutical Co., Ltd., and K. Endo Employee of Chugai Pharmaceutical Co., Ltd. doi:10.1016/j.bone.2012.02.184
NIOP05 / PP212 Strontium ranelate fully prevents alteration of bone mechanical properties in response to cyclical loading P. Ammann⁎, R. Rizzoli Division of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland Abstract: Microarchitecture and intrinsic bone tissue properties contribute independently and significantly to the improvement of bone strength induced by strontium ranelate (SrRan) treatment as evaluated by μCT-based finite element analysis. How improvement of bone material quality by SrRan could influence bone mechanical properties is unknown. One hypothesis is that SrRan present in bone tissue could prevent the formation of micro cracks and/or their propagation, thus influencing the capacity of bones to cumulate micro-damage. We investigated whether SrRan could prevent alteration of bone mechanical properties in response to fatigue at the level of vertebrae. Vertebrae of intact female rats treated over 8 weeks (5/7 days) with SrRan at a dose-level of 625 mg/kg (corresponding approximately to the human SrRan exposure) or with a vehicle (control) were cyclically loaded in axial compression under a controlled load for 100 cycles. The selected peak load corresponded to 5% of the adjacent vertebra maximal load, thus in the domain of elastic deformation. This peak load was determined to induce alteration of post Yield load without inducing fracture. The vertebrae were then loaded to failure. We compared the load/displacement curve of the cyclically loaded vertebra (L4) to the adjacent one (L3), not cyclically loaded. Bone mechanical properties including post-yield load and deflection, characterizing post yield behaviour, were investigated. Unloaded control (n = 9)
Fatigue control (n = 9)
Unloaded SrRan (n = 8)
Fatigue SrRan (n = 8)
Post Yield load (N) 19.81 ± 3.38 11.80 ± 2.03⁎ 18.42 ± 4.00 18.78 ± 3.71 Post Yield deflection 0.067 ± 0.007 0.073 ± 0.017 0.063 ± 0.011 0.072 ± 0.012 (mm) ⁎ p b 0.05 vs. unloaded control adjacent vertebra (mean ± SEM). Maximal load was 267 ± 19 and 233 ± 20 (N) in unloaded SrRan and control groups, respectively. Cyclic loading induced a deterioration of post Yield load (− 40%) in control rats. This effect was fully prevented in SrRan-treated rats. The Post Yield deflection was unaffected in either group. These results indicate that fatigue-induced alterations of post yield behaviour were fully prevented by SrRan treatment. They also suggest that bone micro-damages could be less prominent in SrRan-treated rats (histological analysis in progress). In conclusion, the improvement of bone intrinsic mechanical properties by SrRan could
Abstracts influence the response to cyclical loading (fatigue) and thus represents an original mechanism of action. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: P. Ammann grant/research support from Servier, Novartis, Amgen, Nestlé and Galapagos, R. Rizzoli grant/research support from Merck Sharp and Dome, Eli Lilly, Amgen, Wyeth, Novartis, Servier, Nycomed, Nestlé, Danone. doi:10.1016/j.bone.2012.02.185
NIOP06/PP066 Regulation of osteoclast formation by toll-like receptors 2 and 5 A. Kassema,⁎, P. Lunberga, C. Lindholmb, P. Souzac, U. Lernera a Molecular Periodontology, UMEÅ UNIVERSITY, Umeå, Sweden b Centre for Bone and Arthritis Research, Sahlgrenska Academy at University of Gothenburg, Gothenberg, Sweden c Department of Physiology and Pathology, University of São Paulo State, Araraquara, Brazil Abstract: Infections within or in the vicinity of the skeleton induce osteolytic diseases such as periodontitis, septic arthritis, osteomyelitis, peri-implantitis. Although host production of osteotropic cytokines is crucial, the precise mechanism by which pathogen associated molecular patterns induce osteoclastogenesis and bone loss is not fully understood. Recognition of these patterns by members of the toll-like receptor (TLR) family may be important. We have studied how activation of TLR2 and 5 affects bone resorption, osteoblasts and osteoclast progenitors. Activation of TLR2/1 or TLR2/6 dimers with either LPS from P. gingivalis, heat killed Listeria monocytogenes (HKLM), Pam2CSK4 (ligand for TLR2/6), Pam3CSK4 (ligand for TLR2/1) and FSL1 increased the release of 45Ca from mouse calvarial bones. This response was associated with increased expression of the osteoclastic genes Acp5, Ctsk and Oscar, as well as of C-Fos mRNA, enhanced degradation of bone matrix as assessed by release of the collagen fragment CTX and increased number of cathepsin K+ osteoclasts. TLR2 agonists also increased the mRNA and protein expression of RANKL without affecting OPG mRNA and protein. Recombinant OPG inhibited 45Ca release triggered by TLR2 ligands. All TLR2 agonists increased the mRNA expression of Il-1b, Tnf-a, Il-6 and Cox-2. Anti-IL-1b and anti-TNFa did not affect LPS Pg induced Rankl mRNA, whereas an inhibitor of prostaglandin biosynthesis impaired LPS Pg induced Rankl mRNA by 50%, but still causing a considerable enhancement of Rankl mRNA. Pam2CSK4 induced 45Ca release was unaffected by indomethacin. Osteoblasts isolated from the periosteum of calvarial bones expressed TLR1, 2, 5 and 6. LPS Pg, HKLM, FSL1, Pam2CSK4 Pam3CSK4 induced Rankl mRNA, without affecting Opg mRNA in isolated osteoblasts. Although TLR2 agonists did not affect RANKL stimulated 45Ca release in calvarial bones, all TLR2 agonists robustly inhibited RANKL stimulated osteoclast formation in mouse bone marrow macrophage cultures. Activation of TLR5 by ST-FLA (Flagellin from S. typhimurim) also enhanced 45Ca release and increased Rankl mRNA in calvarial bones without affecting Opg mRNA, a response seen also in calvarial osteoblasts. These data show that TLR2- and TLR5-signaling stimulates periosteal osteoclast formation and bone resorption by enhancing RANKL/OPG ratio in osteoblasts, but TLR2 agonists inhibit RANKL signaling in early osteoclast progenitor cells in bone marrow. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.186
NIOP07/PP053 Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on Oncostatin M signaling P. Guiharda,b,⁎, Y. Dangerc, B. Brounaisa,b, E. Davida,b, R. Briona,b, J. Delecrind, C. Richardse, S. Chevalierc, F. Redinia,b, D. Heymanna,b, H. Gascanc, F. Blancharda,b a U957, INSERM, France b Université de Nantes, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, EA3822, Nantes, France c U564, INSERM, Angers, France d service d'Othopédie-Traumatologie, CHU de Nantes, Nantes, France e Center for Gene Therapeutics, McMaster University, Hamilton, Canada Abstract: Bone resorption by osteoclasts and bone formation by osteoblasts are tight coupled processes implicating factors in the TNF, bone morphogenetic protein
S67
and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating osteoblast-dependent bone formation but the coupling factors were not identified. Using a high throughput approach, we identified Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages. We uncovered the role of this factor as an inducer of C/EBP beta (CCAAT-enhancer-binding protein beta), Cbfa1 and alkaline phosphatase expression, osteoblast differentiation and matrix mineralization from human mesenchymal stem cells (MSC) while inhibiting adipogenesis. Upon toll-like receptors (TLRs) activation by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA against OSM, OSM receptor subunits gp130 and OSMR or to the downstream transcription factor STAT3. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines had also a potent role in bone formation induced by monocytes/macrophages and TLRs activation: IL-6 and Leukemia inhibitory factor. We propose that during bone inflammation, infection or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies. This work was supported by Inserm, le Ministère de la Recherche, and La Ligue Contre le Cancer (comité Grand Ouest). P.G. is a recipient from a fellowship from le Ministère de la Recherche. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.187
NIOP08/PP061 P2Y2 receptor overexpression results in decreased bone formation S. Syberga,⁎, C. Agcab, N. Wangc, S. Petersena, A. Gartlandc, P. Schwarza,d, N.R. Jørgensena, Y. Agcab a Research Center of Ageing and Osteoporosis, Deps. Clinical Biochemistry adn Medicine, Glostrup Hospital, Glostrup, Denmark b College of Veterinary Medicine, University of Missouri, Colombia, USA c Department of Human Metabolism, University of Sheffield, Sheffield, UK d Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Abstract: Nucleotides such as adenosine triphosphate (ATP) are released from bone cells, and subsequently act on P2 purinergic receptors. The P2Y2 subtype is a G-protein coupled receptor expressed in osteoblasts and osteoclasts. In vitro activation of the receptor nucleotides results in inhibition of alkaline phosphatase (ALP) production and matrix mineralization. The aim of this study was therefore to investigate whether overexpression of the P2Y2 receptor affects bone. Wildtype (WT) and P2Y2 overexpressing (P2Y2R) female nullipara rats were used in the whole study. Bones from three-month old calcein-labelled rats were analysed (Piximus densitometer, SkyScan μCT, and bone histomorphometry). Serum was collected for bone marker analysis. Preosteoblasts were isolated from the calvaria of two day old rats for in vitro analysis of osteoblasts from the two genotypes. Bone mineral density (BMD) was reduced in P2Y2R compared to WT and μCT showed reduced trabecular thickness in the P2Y2R 0.057 ± 0.0005 mm vs. 0.059 ± 0.0005 mm. TRAP-5b was decreased in P2Y2R 2.1 ± 0.2U vs. 1.5 ± 0.1U (p b 0.01). This was confirmed by histomorphometry, where P2Y2R showed increased erosive surfaces with ES/BS% of 18.6 ± 0.5% vs. 16.9 ± 0.5% (p b 0.05). In contrast, the number of actively forming surfaces (MS/BS %) was decreased in the P2Y2R 56.9 ± 0.5% vs. 60.6 ± 1.0% (p b 0.01). Fewer osteoblasts developed in calvarial cultures in P2Y2R rats vs. WT. No differences in proliferation could be detected in mature osteoblasts (p = 0.619). However the ability to form mineralized matrix was 43% lower in the osteoblasts from P2Y2R rats (p = 0.002). In P2Y2R osteoblast cultures ALP was 40% reduced compared to WT (p = 0.001). Further analysis will reveal if P2Y2R have increased resorption in vitro as seen in vivo. In conclusion, P2Y2 receptor overexpression in rats results in decreased bone mineral density, partially due to not only increased bone resorption, but also decreased formation (detected by serum bone markers and bone histomorphometry). This is consistent with previously published in vitro effects of P2Y2 receptor activation in osteoblasts. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.188
Abstracts
a
Department of Exercise Sciences/Multidisciplinary Osteoporosis Research Program, University of Toronto/Women's College Hospital, Toronto, Canada b Quantum Medical Metrics LLC, USA c Department of Electrical and Computer Engineering, Johns Hopkins University, Baltimore, USA d Department of Medicine/Multidisciplinary Osteoporosis Research Program, University of Toronto/Women's College Hospital, Toronto, Canada e Department of Medicine, St. Joseph's Healthcare/McMaster University, Hamilton, Canada f Centre de Recherche du CHUQ, Laval University, Quebec City, Canada Abstract: The function of skeletal adaptation to mechanical load (modeling) is to adjust the amount and distribution of bone tissue (geometry) and perhaps material elastic properties, so that strains experienced within the bone are kept within certain physiological limits. Genetic, environmental or hormonal factors may cause heterogeneity in load response (mechanosensitivity), such that equivalent loads would generate higher or lower strains. Individuals who have reduced mechanosensitivity should require higher strains to generate an adaptive response, consequently have weaker bones and fracture earlier and more frequently throughout their life. The purpose of the current study was to determine if stresses (proportional to strains) at the femoral neck under equivalent physiologic loads were higher in women whose first fracture occurred early in life, than in women whose first fracture occurred late in life. We studied 3197 women between the ages of 25 and 92 who participated in the Canadian Multicentre Osteoporosis Study (CaMos) and who had available Hip Structure Analysis (HSA) data from baseline dual energy X-ray absorptiometry (DXA) scans. Women were categorized into 3 groups based on their age at first fracture (b 50 years, between 50–64 years and ≥65 years). We computed stress (megapascals = MPa) at the infero-medial margin of the femoral neck in a one-legged stance mode using a 2-D engineering beam analysis incorporating dimensions and geometry from DXA scans using the HSA method. We also assessed geometry parameters from the HSA analysis including: narrow neck (NN) bone mineral density (BMD), NN cross-sectional area (CSA) and NN section modulus (Z; an index of bending strength). We used general linear models (SAS 9.1) to determine associations between stance stress, geometry parameters and age at first fracture based on fracture groupings. We found that women whose first fracture was early in life (b50 years) had higher stress (10.35 ± 2.19 vs. 10.00 ± 1.99 MPa; p = 0.0468), lower NN BMD (0.757 ± 0.15 vs. 0.784 ± 0.16 g/cm2; p = 0.0306), lower NN CSA (1.895 ± 0.38 vs. 1.997 ± 0.42 cm2; p = 0.0013) and lower NN Z (0.912 ± 0.22 vs. 0.966 ± 0.25 cm3; p = 0.0024) than women whose first fracture was late in life (≥ 65 years). These findings provide evidence of heterogeneity in load response and suggest an important role for modeling in the pathogenesis of bone fragility in women. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: C. Hamilton: none declared, T. Beck Shareholder of Hologic Inc., A. Khaled: none declared, S. Jamal Advisory Board Membership of Novartis, Amgen, Warner-Chilcott, Consulting fees from Novartis, Warner-Chilcott, Genzyme, Shire, Speaker Fees from Novartis, Amgen, Warner-Chilcott, Genzyme, Shire, J. Adachi Grant / Research Support from Amgen, Bristol-Myers Squibb, Eli Lilly, Merck, Novartis, Pfizer, Procter & Gamble, Sanofi Aventis, Roche Warner-Chilcott, Consulting fees from Amgen, Eli Lilly, Glaxo Smith Kline, Merck, Novartis, Pfizer, Procter & Gamble, Roche, Sanofi Aventis, Warner-Chilcott, Speaker Fees from Amgen, Eli Lilly, GlaxoSmithKline, Merck, Novartis, Pfizer, Procter & Gamble, Roche, Sanofi Aventis, Warner-Chilcott, J. Brown Grant / Research Support from Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, Sanofi-Aventis, Servier, Warner-Chilcott, Speakers Bureau with Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, SanofiAventis, Servier, Warner-Chilcott, Consulting fees from Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, Sanofi-Aventis, Servier, WarnerChilcott, Speaker Fees from Abbott, Amgen, Bristol Myers Squibb, Eil Lilly, Merck, Novartis, Pfizer, Rocher, Sanofi-Aventis, Servier, Warner-Chilcott, K. Davison Advisory Board Membership of Amgen, Servier, Novartis, Consulting fees from Merck, Amgen, Novartis, Servier, Speaker Fees from Merck, Warner-Chilcott, Amgen, Novartis, Servier.
doi:10.1016/j.bone.2012.02.183
NIOP04 / PP438 Active vitamin D3 administration improves locomotive ability of mice with rotarod training S. Sakai⁎, M. Suzuki, K. Tanaka, M. Mihara, K. Endo Product Research Department, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan Abstract: Active vitamin D3 improves bone mineral density and bone strength. Moreover, some studies have found that vitamin D3 decreases the risk of falls. However, we have very little knowledge about the relationship between active vitamin D3 and peripheral nerve function, which is known to decline with age. In this study, we used vitamin D receptor knockout (VDR KO) mice to investigate the effect of VDR
signaling on motor function. In addition, we evaluated the effect of a new antiosteoporotic medicine, eldecalcitol (ED-71), an active vitamin D3 analog, on enhancing locomotive ability in mice with rotarod training. Wild type (WT) and VDR KO mice were trained on a rotarod 5 days a week (15 rpm, 5 min, 3 times/day) for 4 weeks, and locomotive performance was then measured by an accelerating rotarod test. Four-week training led to an increased duration on the rotarod in the WT mice but not in the VDR KO mice, suggesting that VDR signaling is essential for the improvement of motor function. Histological observations of sciatic nerve cross sections revealed that the axon diameter was smaller in VDR KO mice than in WT mice. Additionally, the distribution of acetylcholine receptor on the extensor digitorum longus muscle was not as broad and the pattern of distribution was simpler in the VDR KO mice. Furthermore, active vitamin D3 dose-dependently upregulated mRNA expression of insulin-like growth factor-I (IGF-I) and myelin basic protein (MBP) in Schwann cells isolated from neonatal rats. We assessed the effect of ED-71 administration on locomotive ability of mice during rotarod training. Nine-week-old male C57BL mice either receiving or not receiving orally administered ED-71 were trained on a rotarod following the same training regimen as above for 2 weeks. The locomotive ability of trained mice receiving ED-71 was superior than that of those not receiving ED-71 treatment. In conclusion, VDR signaling plays a critical role in upregulating motor function in the rotarod training model. Stimulation with active vitamin D3 increased mRNA expression of IGF-I and MBP in Schwann cells in vitro. These results suggest that the improved motor function observed following administration of ED-71 may be via the induction of IGF-I and MBP from peripheral Schwann cells. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: S. Sakai Employee of Chugai Pharmaceutical Co., Ltd., M. Suzuki Employee of Chugai Pharmaceutical Co., Ltd., K. Tanaka Employee of Chugai Pharmaceutical Co., Ltd., M. Mihara Employee of Chugai Pharmaceutical Co., Ltd., and K. Endo Employee of Chugai Pharmaceutical Co., Ltd. doi:10.1016/j.bone.2012.02.184
NIOP05 / PP212 Strontium ranelate fully prevents alteration of bone mechanical properties in response to cyclical loading P. Ammann⁎, R. Rizzoli Division of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland Abstract: Microarchitecture and intrinsic bone tissue properties contribute independently and significantly to the improvement of bone strength induced by strontium ranelate (SrRan) treatment as evaluated by μCT-based finite element analysis. How improvement of bone material quality by SrRan could influence bone mechanical properties is unknown. One hypothesis is that SrRan present in bone tissue could prevent the formation of micro cracks and/or their propagation, thus influencing the capacity of bones to cumulate micro-damage. We investigated whether SrRan could prevent alteration of bone mechanical properties in response to fatigue at the level of vertebrae. Vertebrae of intact female rats treated over 8 weeks (5/7 days) with SrRan at a dose-level of 625 mg/kg (corresponding approximately to the human SrRan exposure) or with a vehicle (control) were cyclically loaded in axial compression under a controlled load for 100 cycles. The selected peak load corresponded to 5% of the adjacent vertebra maximal load, thus in the domain of elastic deformation. This peak load was determined to induce alteration of post Yield load without inducing fracture. The vertebrae were then loaded to failure. We compared the load/displacement curve of the cyclically loaded vertebra (L4) to the adjacent one (L3), not cyclically loaded. Bone mechanical properties including post-yield load and deflection, characterizing post yield behaviour, were investigated. Unloaded control (n = 9)
Fatigue control (n = 9)
Unloaded SrRan (n = 8)
Fatigue SrRan (n = 8)
Post Yield load (N) 19.81 ± 3.38 11.80 ± 2.03⁎ 18.42 ± 4.00 18.78 ± 3.71 Post Yield deflection 0.067 ± 0.007 0.073 ± 0.017 0.063 ± 0.011 0.072 ± 0.012 (mm) ⁎ p b 0.05 vs. unloaded control adjacent vertebra (mean ± SEM). Maximal load was 267 ± 19 and 233 ± 20 (N) in unloaded SrRan and control groups, respectively. Cyclic loading induced a deterioration of post Yield load (− 40%) in control rats. This effect was fully prevented in SrRan-treated rats. The Post Yield deflection was unaffected in either group. These results indicate that fatigue-induced alterations of post yield behaviour were fully prevented by SrRan treatment. They also suggest that bone micro-damages could be less prominent in SrRan-treated rats (histological analysis in progress). In conclusion, the improvement of bone intrinsic mechanical properties by SrRan could
Abstracts influence the response to cyclical loading (fatigue) and thus represents an original mechanism of action. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: P. Ammann grant/research support from Servier, Novartis, Amgen, Nestlé and Galapagos, R. Rizzoli grant/research support from Merck Sharp and Dome, Eli Lilly, Amgen, Wyeth, Novartis, Servier, Nycomed, Nestlé, Danone. doi:10.1016/j.bone.2012.02.185
NIOP06/PP066 Regulation of osteoclast formation by toll-like receptors 2 and 5 A. Kassema,⁎, P. Lunberga, C. Lindholmb, P. Souzac, U. Lernera a Molecular Periodontology, UMEÅ UNIVERSITY, Umeå, Sweden b Centre for Bone and Arthritis Research, Sahlgrenska Academy at University of Gothenburg, Gothenberg, Sweden c Department of Physiology and Pathology, University of São Paulo State, Araraquara, Brazil Abstract: Infections within or in the vicinity of the skeleton induce osteolytic diseases such as periodontitis, septic arthritis, osteomyelitis, peri-implantitis. Although host production of osteotropic cytokines is crucial, the precise mechanism by which pathogen associated molecular patterns induce osteoclastogenesis and bone loss is not fully understood. Recognition of these patterns by members of the toll-like receptor (TLR) family may be important. We have studied how activation of TLR2 and 5 affects bone resorption, osteoblasts and osteoclast progenitors. Activation of TLR2/1 or TLR2/6 dimers with either LPS from P. gingivalis, heat killed Listeria monocytogenes (HKLM), Pam2CSK4 (ligand for TLR2/6), Pam3CSK4 (ligand for TLR2/1) and FSL1 increased the release of 45Ca from mouse calvarial bones. This response was associated with increased expression of the osteoclastic genes Acp5, Ctsk and Oscar, as well as of C-Fos mRNA, enhanced degradation of bone matrix as assessed by release of the collagen fragment CTX and increased number of cathepsin K+ osteoclasts. TLR2 agonists also increased the mRNA and protein expression of RANKL without affecting OPG mRNA and protein. Recombinant OPG inhibited 45Ca release triggered by TLR2 ligands. All TLR2 agonists increased the mRNA expression of Il-1b, Tnf-a, Il-6 and Cox-2. Anti-IL-1b and anti-TNFa did not affect LPS Pg induced Rankl mRNA, whereas an inhibitor of prostaglandin biosynthesis impaired LPS Pg induced Rankl mRNA by 50%, but still causing a considerable enhancement of Rankl mRNA. Pam2CSK4 induced 45Ca release was unaffected by indomethacin. Osteoblasts isolated from the periosteum of calvarial bones expressed TLR1, 2, 5 and 6. LPS Pg, HKLM, FSL1, Pam2CSK4 Pam3CSK4 induced Rankl mRNA, without affecting Opg mRNA in isolated osteoblasts. Although TLR2 agonists did not affect RANKL stimulated 45Ca release in calvarial bones, all TLR2 agonists robustly inhibited RANKL stimulated osteoclast formation in mouse bone marrow macrophage cultures. Activation of TLR5 by ST-FLA (Flagellin from S. typhimurim) also enhanced 45Ca release and increased Rankl mRNA in calvarial bones without affecting Opg mRNA, a response seen also in calvarial osteoblasts. These data show that TLR2- and TLR5-signaling stimulates periosteal osteoclast formation and bone resorption by enhancing RANKL/OPG ratio in osteoblasts, but TLR2 agonists inhibit RANKL signaling in early osteoclast progenitor cells in bone marrow. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.186
NIOP07/PP053 Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on Oncostatin M signaling P. Guiharda,b,⁎, Y. Dangerc, B. Brounaisa,b, E. Davida,b, R. Briona,b, J. Delecrind, C. Richardse, S. Chevalierc, F. Redinia,b, D. Heymanna,b, H. Gascanc, F. Blancharda,b a U957, INSERM, France b Université de Nantes, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, EA3822, Nantes, France c U564, INSERM, Angers, France d service d'Othopédie-Traumatologie, CHU de Nantes, Nantes, France e Center for Gene Therapeutics, McMaster University, Hamilton, Canada Abstract: Bone resorption by osteoclasts and bone formation by osteoblasts are tight coupled processes implicating factors in the TNF, bone morphogenetic protein
S67
and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating osteoblast-dependent bone formation but the coupling factors were not identified. Using a high throughput approach, we identified Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages. We uncovered the role of this factor as an inducer of C/EBP beta (CCAAT-enhancer-binding protein beta), Cbfa1 and alkaline phosphatase expression, osteoblast differentiation and matrix mineralization from human mesenchymal stem cells (MSC) while inhibiting adipogenesis. Upon toll-like receptors (TLRs) activation by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA against OSM, OSM receptor subunits gp130 and OSMR or to the downstream transcription factor STAT3. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines had also a potent role in bone formation induced by monocytes/macrophages and TLRs activation: IL-6 and Leukemia inhibitory factor. We propose that during bone inflammation, infection or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies. This work was supported by Inserm, le Ministère de la Recherche, and La Ligue Contre le Cancer (comité Grand Ouest). P.G. is a recipient from a fellowship from le Ministère de la Recherche. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.187
NIOP08/PP061 P2Y2 receptor overexpression results in decreased bone formation S. Syberga,⁎, C. Agcab, N. Wangc, S. Petersena, A. Gartlandc, P. Schwarza,d, N.R. Jørgensena, Y. Agcab a Research Center of Ageing and Osteoporosis, Deps. Clinical Biochemistry adn Medicine, Glostrup Hospital, Glostrup, Denmark b College of Veterinary Medicine, University of Missouri, Colombia, USA c Department of Human Metabolism, University of Sheffield, Sheffield, UK d Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Abstract: Nucleotides such as adenosine triphosphate (ATP) are released from bone cells, and subsequently act on P2 purinergic receptors. The P2Y2 subtype is a G-protein coupled receptor expressed in osteoblasts and osteoclasts. In vitro activation of the receptor nucleotides results in inhibition of alkaline phosphatase (ALP) production and matrix mineralization. The aim of this study was therefore to investigate whether overexpression of the P2Y2 receptor affects bone. Wildtype (WT) and P2Y2 overexpressing (P2Y2R) female nullipara rats were used in the whole study. Bones from three-month old calcein-labelled rats were analysed (Piximus densitometer, SkyScan μCT, and bone histomorphometry). Serum was collected for bone marker analysis. Preosteoblasts were isolated from the calvaria of two day old rats for in vitro analysis of osteoblasts from the two genotypes. Bone mineral density (BMD) was reduced in P2Y2R compared to WT and μCT showed reduced trabecular thickness in the P2Y2R 0.057 ± 0.0005 mm vs. 0.059 ± 0.0005 mm. TRAP-5b was decreased in P2Y2R 2.1 ± 0.2U vs. 1.5 ± 0.1U (p b 0.01). This was confirmed by histomorphometry, where P2Y2R showed increased erosive surfaces with ES/BS% of 18.6 ± 0.5% vs. 16.9 ± 0.5% (p b 0.05). In contrast, the number of actively forming surfaces (MS/BS %) was decreased in the P2Y2R 56.9 ± 0.5% vs. 60.6 ± 1.0% (p b 0.01). Fewer osteoblasts developed in calvarial cultures in P2Y2R rats vs. WT. No differences in proliferation could be detected in mature osteoblasts (p = 0.619). However the ability to form mineralized matrix was 43% lower in the osteoblasts from P2Y2R rats (p = 0.002). In P2Y2R osteoblast cultures ALP was 40% reduced compared to WT (p = 0.001). Further analysis will reveal if P2Y2R have increased resorption in vitro as seen in vivo. In conclusion, P2Y2 receptor overexpression in rats results in decreased bone mineral density, partially due to not only increased bone resorption, but also decreased formation (detected by serum bone markers and bone histomorphometry). This is consistent with previously published in vitro effects of P2Y2 receptor activation in osteoblasts. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.188