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Reduced physical activity corresponds with greater bone loss at the trabecular than the cortical bone sites: A 12-year prospective cohort study of young Swedish men
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Abstracts / Bone 44 (2009) S253–S338
polarization as a function of animal age perpendicularly to the longitudinal direction of the diaphysis and a smaller one in the metaphysis of mice femora (based on the variation of the amide I integrated area). This may be a reflection of the varying tissue age within the same specimen. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.471
P046 Strains in bone and tooth via high energy X-ray scattering S.R. Stocka,*, J.D. Almerb a Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine Northwestern Univ., Chicago, IL, USA b Advanced Photon Source, Argonne National Lab, Argonne, IL, USA Background/aims: High energy synchrotron X-ray scattering allows quantification of internal strains within bone and tooth samples. The aim is to improve understanding of apatite and collagen function in these composites by repeated observation of the same bulk specimen. Methods: Wide angle and small angle X-ray scattering was used to quantify strain distributions in bone and tooth at the Advanced Photon Source using variation of apatite d-spacing and collagen D-period in high energy (>60 keV) patterns. Millimeter thick specimens were examined with transmission under in situ load and under de-/re-hydrating environments. Results: Four results obtained with microbeams illustrate the approach. Substantial strain gradients were observed across a rat tibia loaded in compression (figure). Strain maps across the dentinoenamel junction in a block of bovine incisor revealed, under compression, that strain peaks on the enamel side and dips on the dentin side of the junction. During drying, residual strains exhibited diffusion controlled kinetics and became more tensile by 2500 < \mu \epsi < 4000 in bovine bone. Methods for 3D scattering tomography were developed and applied. Conclusions: Perturbations of collagen other than dehydration should expand what can be learned of its interaction with the mineral phase. It should be possible to extend the non-invasive strain mapping approach to observe how assemblies of bone interact, how load is distributed over intact teeth, perhaps in in vivo animal models.
P047 Development of a new ELISA for serum periostin: Growth-related changes and effects of bisphosphonate in mice S. Contiéa,*, N. Voorzanger-Rousselota, J. Litvinb, N. Bonnetc, S. Ferraric, S.J. Conwayd, P. Clézardine, P. Garneroa a Biochemical Markers, CCBR-Synarc, Lyon, France b Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, USA c Service of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland d Cardiovascular Development Group, Herman B Wells Center for Pediatrics Research, Indiana University School of Medicine, Indianapolis, USA e Research Unit 664, INSERM, Lyon, France Background: The non-collagenous protein periostin (POSTN) comprises several (4 in mouse, 6 in humans) isoforms resulting from alternative splicing in the C-terminal region. It is highly expressed in the periosteum and in bone marrow stromal cells. Recent studies in POSTN−/− mice suggest a role for POSTN in regulating bone formation and bone mass. The aim of our study was to develop a specific ELISA for serum POSTN and investigate the growth-related and bisphosphonate induced changes on POSTN levels in mice. Methods: The POSTN ELISA is based on a polyclonal antibody raised against the C-Terminal sequence common to all 4 mouse isoforms. Serum POSTN levels were measured in POSTN knockout (KO), heterozygote (HET) and wild-type (WT) C57Bl6/J female mice, in 4-, 6-, 8-, 10- and 12-week old WT Balb/c mice, and in 8-week old ovariectomized Balb/c mice receiving zoledronic acid (n = 9) or placebo (n = 10) for 6 weeks. Established serum biochemical markers of bone formation (osteocalcin and PINP) and bone resorption (TRACP5b) were also evaluated. Results: The intra- and inter-assay coefficients of variation of serum POSTN ELISA were 9.5% and 11.9%, respectively. The mean dilution recovery ranged from 95% to 104%. The detection limit was determined at 1.4 ng/ml. Serum levels of POSTN were 50% lower in HET POSTN−/+ mice than in WT animals and barely detectable in double knock-out mice, demonstrating the specificity of the ELISA. Serum periostin levels decreased with age, with a plateau reached at 8 weeks. A similar pattern was observed for markers of bone formation and resorption, although none of them significantly correlated with POSTN levels. Zoledronic acid induced a reduction of osteocalcin, PINP and TRACP5b by 50%, 47% and 33%, respectively (p < 0.005), contrasting with the absence of effect on serum POSTN levels. Immunohistochemistry showed that periostin localized specifically in the perichondrium, and the staining decreased in older animals. Conclusion: The new ELISA allows accurate and precise measurements of serum POSTN in mice. Serum POSTN is not a marker of bone turnover, but more probably reflects modifications in the growthassociated intramembranous ossification. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.473
Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.472
P048 Reduced physical activity corresponds with greater bone loss at the trabecular than the cortical bone sites: A 12-year prospective cohort study of young Swedish men T.T. Tervoa,*, P. Nordströmb, M. Neoviusc, A. Nordströmd a Surgical and Perioperative Sciences, Sports Medicine, Umeå, Sweden b Department of Community Medicine and Rehabilitation, Geriatrics, Umeå, Sweden c Department of Medicine, Rehabilitation Medicine, Stockholm, Sweden d Rehabilitation and Community Medicine, Rehabilitation Medicine, Umeå, Sweden
Abstracts / Bone 44 (2009) S253–S338
Previous research has been inconclusive as to whether high peak bone mineral density (BMD, g/cm2) resulting from previous physical activity is retained with reduced activity later in life. The aim of this 12-year longitudinal study was to investigate BMD loss at reduced levels of physical activity at both trabecular and cortical bone sites. Three groups with a mean age of 17 years at baseline were investigated: i) 51 athletes who discontinued their active careers during the follow-up period (former athletes), ii) 16 athletes who were active throughout the follow-up period (active athletes), and iii) 25 controls. BMD loss at hip, spine, and pelvis (mainly trabecular bone) was compared to BMD loss at femur, humerus, and legs (mainly cortical bone) during a 12-year followup period. Across the total follow-up period in the total cohort, reduced physical activity was more strongly associated with changes at trabecular BMD sites, i.e. hip, spine, and pelvis (B = 0.008–0.005 g/cm2 per hour physical activity (h), p < 0.001), than at cortical bone sites, i.e. humerus, legs (B = 0.002–0.003 g/ cm2 per h, p < 0.05), and femur (B = 0.02 g/cm2 per h, p > 0.05). At the final follow-up, former athletes showed higher BMD than controls only at the cortical bone sites of the humerus, legs, and femur (difference 0.05–0.10 g/cm2, p < 0.05). In summary, this study indicates that predominantly trabecular bone is lost with reduced physical activity levels in young men. Benefits were still evident at the more cortical humerus, legs, and femur eight years after the discontinuation of an active sports career. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.474
P049 Interrelationships between densitometric, morphometric, and mechanical properties of femur in male Polish Merino sheep W. Krupskia,*, M.R. Tatarab, R. Palczakc, R. Maciejewskid a II Department of Radiology, Medical University of Lublin, Lublin, Poland b Department of Animal Physiology, University of Life Sciences in Lublin, Lublin, Poland c Department of Public Health, University of Information Technology and Management, Rzeszów, Poland d Human Anatomy Department, Medical University of Lublin, Lublin, Poland The aim of the study was to evaluate the interrelationships between parameters assessed with the use of computed tomography technique and mechanical properties of femur in male Polish Merino sheep. Healthy ram lambs (N = 24) were kept under standard rearing conditions to the slaughter age of 6 months of life to obtain femur for analyses. Using computed tomography technique (Somatom Emotion apparatus, Siemens, Germany), volumetric bone mineral density (vBMD) of the trabecular (Td) and cortical bone (Cd), mean volumetric bone mineral density (MvBMD) and total bone volume (Bvol) of femur were evaluated. Geometrical properties such as cross-sectional area (A), second moment of inertia (Ix), mean relative wall thickness (MRWT) and cortical index (CI) were derived on the basis of computed tomography measurements of horizontal and vertical diameters of the femur in the midshaft. Using an INSTRON 3367 apparatus (Instron, USA) and three-point bending test, the mechanical parameters such as maximum elastic strength (Wy) and ultimate strength (Wf) of femur were estimated. Pearson's correlation coefficient (r) was determined for all the investigated variables and P < 0.05 was considered as statistically significant. Obtained results showed positive correlations between Td and Cd. These parameters were positively correlated with bone weight and
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length, Bvol, MvBMD, A and Ix. Volumetric BMD of Cd was positively correlated with Wy and Wf. Total bone volume was positively correlated with bone weight, length, A and Ix. Bone length was positively correlated with bone weight, MvBMD, A, Ix and Wy. Bone weight was positively correlated with A and Ix. Positive correlations were also found between Wy and Wf, A and Ix, as well as MRWT and CI. Negative correlations of MRWT and CI with bone weight, Bvol and Ix were stated. In conclusion, this study showed computed tomography as a precise technique for determination of skeletal system properties that may serve to monitor metabolic response of bone tissue in studies on physiological, nutritional and pharmacological factors influencing skeletal system. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.475
P050 The evolution of size and distribution of apatite mineral crystals during bone fracture healing in sheep Y. Liua,*, I. Manjubalaa, P. Roschgerb, D.R. Eparic, H. Schelld, J. Lienaud, H.J. Baild, G.N. Dudad, P. Fratzla a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany b 4th Medical Department, Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1140 Vienna, Austria c School of Engineering Systems, Institute of Health and Biomedical Innovation (IHBI), Faculty of Built Environment and Engineering, Queensland University of Technology, 4001 Brisbane, Australia d Julius Wolff Institut and Center for Musculoskeletal Surgery, Charité-University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany Mature bone is known to be a very complex tissue with hierarchical structure containing well-organized mineral particles and organic matrix. At a similar scale, the structure properties of the fracture callus together with its development through the healing process are, however, relatively unclear. This study aims at investigating the micro-structural characteristics of the newly formed bony materials over the course of transverse diaphyseal fracture healing in a sheep model treated with a rigid mono-lateral external fixator. Since it is known that mineral phase plays an important role in the mechanical performance of bone, we used scanning small angle X-ray scattering (sSAXS) to reveal structural properties of the mineral crystals within the regenerating callus tissue at different healing stages (2, 3, 6 and 9 weeks). Regions of interest were chosen to be periosteal callus and cortex (at all time points) and, in addition, the fracture gap and the periosteal callus on the height of the osteotomy (6 and 9 weeks, since there was no mineral at earlier time points). Mean mineral thickness (T paramenter) and average degree of mineral alignment (ρ parameter) were deduced from resulting sSAXS patterns with a scanning resolution of 200 μum. 2-D maps of T and ρ in correlation with environmental scanning electron microscopy (ESEM) images reveal a gradient in the mineral particle thickness and show that their orientation in callus follows the trabeculae structure. The combination of sSAXS and ESEM data reveals three very different tissue types, (1) an unmineralized organic tissue, (2) a mineralized but poorly organized woven tissue and (3) mostly lamellar bonetype tissue. From the spatial mapping at different time points it becomes apparent that the transformation from (1) to (2) and from (2) to (3) occur in two sequential “waves” propagating along the cortex towards the fracture gap. At the same time as the callus mineralizes and matures, the size of mineral particles decreases in
Abstracts / Bone 44 (2009) S253–S338
polarization as a function of animal age perpendicularly to the longitudinal direction of the diaphysis and a smaller one in the metaphysis of mice femora (based on the variation of the amide I integrated area). This may be a reflection of the varying tissue age within the same specimen. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.471
P046 Strains in bone and tooth via high energy X-ray scattering S.R. Stocka,*, J.D. Almerb a Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine Northwestern Univ., Chicago, IL, USA b Advanced Photon Source, Argonne National Lab, Argonne, IL, USA Background/aims: High energy synchrotron X-ray scattering allows quantification of internal strains within bone and tooth samples. The aim is to improve understanding of apatite and collagen function in these composites by repeated observation of the same bulk specimen. Methods: Wide angle and small angle X-ray scattering was used to quantify strain distributions in bone and tooth at the Advanced Photon Source using variation of apatite d-spacing and collagen D-period in high energy (>60 keV) patterns. Millimeter thick specimens were examined with transmission under in situ load and under de-/re-hydrating environments. Results: Four results obtained with microbeams illustrate the approach. Substantial strain gradients were observed across a rat tibia loaded in compression (figure). Strain maps across the dentinoenamel junction in a block of bovine incisor revealed, under compression, that strain peaks on the enamel side and dips on the dentin side of the junction. During drying, residual strains exhibited diffusion controlled kinetics and became more tensile by 2500 < \mu \epsi < 4000 in bovine bone. Methods for 3D scattering tomography were developed and applied. Conclusions: Perturbations of collagen other than dehydration should expand what can be learned of its interaction with the mineral phase. It should be possible to extend the non-invasive strain mapping approach to observe how assemblies of bone interact, how load is distributed over intact teeth, perhaps in in vivo animal models.
P047 Development of a new ELISA for serum periostin: Growth-related changes and effects of bisphosphonate in mice S. Contiéa,*, N. Voorzanger-Rousselota, J. Litvinb, N. Bonnetc, S. Ferraric, S.J. Conwayd, P. Clézardine, P. Garneroa a Biochemical Markers, CCBR-Synarc, Lyon, France b Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, USA c Service of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland d Cardiovascular Development Group, Herman B Wells Center for Pediatrics Research, Indiana University School of Medicine, Indianapolis, USA e Research Unit 664, INSERM, Lyon, France Background: The non-collagenous protein periostin (POSTN) comprises several (4 in mouse, 6 in humans) isoforms resulting from alternative splicing in the C-terminal region. It is highly expressed in the periosteum and in bone marrow stromal cells. Recent studies in POSTN−/− mice suggest a role for POSTN in regulating bone formation and bone mass. The aim of our study was to develop a specific ELISA for serum POSTN and investigate the growth-related and bisphosphonate induced changes on POSTN levels in mice. Methods: The POSTN ELISA is based on a polyclonal antibody raised against the C-Terminal sequence common to all 4 mouse isoforms. Serum POSTN levels were measured in POSTN knockout (KO), heterozygote (HET) and wild-type (WT) C57Bl6/J female mice, in 4-, 6-, 8-, 10- and 12-week old WT Balb/c mice, and in 8-week old ovariectomized Balb/c mice receiving zoledronic acid (n = 9) or placebo (n = 10) for 6 weeks. Established serum biochemical markers of bone formation (osteocalcin and PINP) and bone resorption (TRACP5b) were also evaluated. Results: The intra- and inter-assay coefficients of variation of serum POSTN ELISA were 9.5% and 11.9%, respectively. The mean dilution recovery ranged from 95% to 104%. The detection limit was determined at 1.4 ng/ml. Serum levels of POSTN were 50% lower in HET POSTN−/+ mice than in WT animals and barely detectable in double knock-out mice, demonstrating the specificity of the ELISA. Serum periostin levels decreased with age, with a plateau reached at 8 weeks. A similar pattern was observed for markers of bone formation and resorption, although none of them significantly correlated with POSTN levels. Zoledronic acid induced a reduction of osteocalcin, PINP and TRACP5b by 50%, 47% and 33%, respectively (p < 0.005), contrasting with the absence of effect on serum POSTN levels. Immunohistochemistry showed that periostin localized specifically in the perichondrium, and the staining decreased in older animals. Conclusion: The new ELISA allows accurate and precise measurements of serum POSTN in mice. Serum POSTN is not a marker of bone turnover, but more probably reflects modifications in the growthassociated intramembranous ossification. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.473
Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.472
P048 Reduced physical activity corresponds with greater bone loss at the trabecular than the cortical bone sites: A 12-year prospective cohort study of young Swedish men T.T. Tervoa,*, P. Nordströmb, M. Neoviusc, A. Nordströmd a Surgical and Perioperative Sciences, Sports Medicine, Umeå, Sweden b Department of Community Medicine and Rehabilitation, Geriatrics, Umeå, Sweden c Department of Medicine, Rehabilitation Medicine, Stockholm, Sweden d Rehabilitation and Community Medicine, Rehabilitation Medicine, Umeå, Sweden
Abstracts / Bone 44 (2009) S253–S338
Previous research has been inconclusive as to whether high peak bone mineral density (BMD, g/cm2) resulting from previous physical activity is retained with reduced activity later in life. The aim of this 12-year longitudinal study was to investigate BMD loss at reduced levels of physical activity at both trabecular and cortical bone sites. Three groups with a mean age of 17 years at baseline were investigated: i) 51 athletes who discontinued their active careers during the follow-up period (former athletes), ii) 16 athletes who were active throughout the follow-up period (active athletes), and iii) 25 controls. BMD loss at hip, spine, and pelvis (mainly trabecular bone) was compared to BMD loss at femur, humerus, and legs (mainly cortical bone) during a 12-year followup period. Across the total follow-up period in the total cohort, reduced physical activity was more strongly associated with changes at trabecular BMD sites, i.e. hip, spine, and pelvis (B = 0.008–0.005 g/cm2 per hour physical activity (h), p < 0.001), than at cortical bone sites, i.e. humerus, legs (B = 0.002–0.003 g/ cm2 per h, p < 0.05), and femur (B = 0.02 g/cm2 per h, p > 0.05). At the final follow-up, former athletes showed higher BMD than controls only at the cortical bone sites of the humerus, legs, and femur (difference 0.05–0.10 g/cm2, p < 0.05). In summary, this study indicates that predominantly trabecular bone is lost with reduced physical activity levels in young men. Benefits were still evident at the more cortical humerus, legs, and femur eight years after the discontinuation of an active sports career. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.474
P049 Interrelationships between densitometric, morphometric, and mechanical properties of femur in male Polish Merino sheep W. Krupskia,*, M.R. Tatarab, R. Palczakc, R. Maciejewskid a II Department of Radiology, Medical University of Lublin, Lublin, Poland b Department of Animal Physiology, University of Life Sciences in Lublin, Lublin, Poland c Department of Public Health, University of Information Technology and Management, Rzeszów, Poland d Human Anatomy Department, Medical University of Lublin, Lublin, Poland The aim of the study was to evaluate the interrelationships between parameters assessed with the use of computed tomography technique and mechanical properties of femur in male Polish Merino sheep. Healthy ram lambs (N = 24) were kept under standard rearing conditions to the slaughter age of 6 months of life to obtain femur for analyses. Using computed tomography technique (Somatom Emotion apparatus, Siemens, Germany), volumetric bone mineral density (vBMD) of the trabecular (Td) and cortical bone (Cd), mean volumetric bone mineral density (MvBMD) and total bone volume (Bvol) of femur were evaluated. Geometrical properties such as cross-sectional area (A), second moment of inertia (Ix), mean relative wall thickness (MRWT) and cortical index (CI) were derived on the basis of computed tomography measurements of horizontal and vertical diameters of the femur in the midshaft. Using an INSTRON 3367 apparatus (Instron, USA) and three-point bending test, the mechanical parameters such as maximum elastic strength (Wy) and ultimate strength (Wf) of femur were estimated. Pearson's correlation coefficient (r) was determined for all the investigated variables and P < 0.05 was considered as statistically significant. Obtained results showed positive correlations between Td and Cd. These parameters were positively correlated with bone weight and
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length, Bvol, MvBMD, A and Ix. Volumetric BMD of Cd was positively correlated with Wy and Wf. Total bone volume was positively correlated with bone weight, length, A and Ix. Bone length was positively correlated with bone weight, MvBMD, A, Ix and Wy. Bone weight was positively correlated with A and Ix. Positive correlations were also found between Wy and Wf, A and Ix, as well as MRWT and CI. Negative correlations of MRWT and CI with bone weight, Bvol and Ix were stated. In conclusion, this study showed computed tomography as a precise technique for determination of skeletal system properties that may serve to monitor metabolic response of bone tissue in studies on physiological, nutritional and pharmacological factors influencing skeletal system. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.475
P050 The evolution of size and distribution of apatite mineral crystals during bone fracture healing in sheep Y. Liua,*, I. Manjubalaa, P. Roschgerb, D.R. Eparic, H. Schelld, J. Lienaud, H.J. Baild, G.N. Dudad, P. Fratzla a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany b 4th Medical Department, Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1140 Vienna, Austria c School of Engineering Systems, Institute of Health and Biomedical Innovation (IHBI), Faculty of Built Environment and Engineering, Queensland University of Technology, 4001 Brisbane, Australia d Julius Wolff Institut and Center for Musculoskeletal Surgery, Charité-University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany Mature bone is known to be a very complex tissue with hierarchical structure containing well-organized mineral particles and organic matrix. At a similar scale, the structure properties of the fracture callus together with its development through the healing process are, however, relatively unclear. This study aims at investigating the micro-structural characteristics of the newly formed bony materials over the course of transverse diaphyseal fracture healing in a sheep model treated with a rigid mono-lateral external fixator. Since it is known that mineral phase plays an important role in the mechanical performance of bone, we used scanning small angle X-ray scattering (sSAXS) to reveal structural properties of the mineral crystals within the regenerating callus tissue at different healing stages (2, 3, 6 and 9 weeks). Regions of interest were chosen to be periosteal callus and cortex (at all time points) and, in addition, the fracture gap and the periosteal callus on the height of the osteotomy (6 and 9 weeks, since there was no mineral at earlier time points). Mean mineral thickness (T paramenter) and average degree of mineral alignment (ρ parameter) were deduced from resulting sSAXS patterns with a scanning resolution of 200 μum. 2-D maps of T and ρ in correlation with environmental scanning electron microscopy (ESEM) images reveal a gradient in the mineral particle thickness and show that their orientation in callus follows the trabeculae structure. The combination of sSAXS and ESEM data reveals three very different tissue types, (1) an unmineralized organic tissue, (2) a mineralized but poorly organized woven tissue and (3) mostly lamellar bonetype tissue. From the spatial mapping at different time points it becomes apparent that the transformation from (1) to (2) and from (2) to (3) occur in two sequential “waves” propagating along the cortex towards the fracture gap. At the same time as the callus mineralizes and matures, the size of mineral particles decreases in