- Email: [email protected]
Femoral bone matrix mineralization is not altered in a rat model of type 2 diabetes mellitus
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Abstracts / Bone 48 (2011) S173–S186
properties in sheep. This study was performed on Polish Merino Sheep that were kept under standard rearing conditions to slaughter at the age of 5 months of life to obtain mandible and teeth for analyses. After initial analysis of weight, length, morphometric, densitometric and mechanical properties of the complete mandible, five teeth (I1-I3, C1 and PM2) were extracted and evaluated individually. Using quantitative computed tomography technique (Somatom Emotion apparatus, Siemens), volumetric bone mineral density (vBMD) and total bone volume (Bvol) of mandible was measured. 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 measurements of horizontal and vertical diameters of cross-section of mandible at 50% length of diastema. Using an INSTRON 3367 apparatus (Instron, USA) and three-point bending test, mechanical parameters such as maximum elastic strength (Wy) and ultimate strength (Wf) of the right and left mandible body were estimated. Furthermore, weight, length, total volume and mean vBMD of teeth was measured. The values of weight, length, Bvol, mean vBMD, vBMD of the cortical bone and calcium hydroxyapatite density of mandible reached 116±6 g, 219±3 mm, 72±cm3, 1.864±0.02 g/cm3, 2.451±0.02 g/cm3 and 1054±26 mgCaHA/mL, respectively. The values of A, Ix, MRWT, CI, Wy and Wf of mandible were 54±2 mm2, 265±21, 0.797±0.02, 44±1, 823±42 N and 1216±106 N, respectively. The values of weight, length, total volume and mean vBMD of teeth were within the ranges of 150±3 mg–783±35 mg, 19±0.3 mm–28±0.5 mm, 113±2 mm3–491± 21 mm3 and 2.947±0.038 g/cm3–3.054±0.034 g/cm3, respectively. In conclusion, this study showed that determination of densitometric, morphometric and mechanical properties of mandible and teeth may serve as a novel attractive model for further investigations on metabolic response of calcified tissues to physiological, pharmacological, nutritional and toxicological factors. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.416
PP257-M Effects of treatment with cyclosporine, FK-506 or rapamycin on femoral biomechanical properties in male adult healthy rats M. Rubert a,⁎, D. Guede b, M. Montero a, J.R. Caeiro c, M. Martín-Fernández a, M. Diaz-Curiel d, C. De la Piedra a a Biochemistry Investigation, Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz, Madrid, Spain b Trabeculae S.L., Technological Park of Galicia, Ourense, Spain c Traumatology and Orthopedic Surgery, University Hospital Complex of Santiago de Compostela, Santiago de Compostela, Spain d Internal Medicine, Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz, Madrid, Spain Abstract: One of the factors which can contribute to the severe bone loss after transplantation is the direct action of immunosuppressants on bone cells. In a previous work we found that tacrolimus (FK-506) administration produced a decrease in femoral (F) and lumbar (L) bone mineral density (BMD). Rapamycin (RAPA) only decreased FBMD and cyclosporine (CsA) did not affect L or FBMD in rats. The aim of this work was to study the effects produced by CsA, FK-506 and RAPA on femoral biomechanical properties in male adult healthy rats. Forty-eight (12/group), 5-month-old male Wistar rats were used. CsA (2 mg/kg/day), FK-506 (3 mg/kg/day), RAPA (1.25 mg/kg/day) or water (0.5 mL/rat/day, CONTROL group) were administered by oral gavage for 3 months. After sacrifice, blood levels of immunosuppressants were determined and the right femur was excised and cleaned for determination of biomechanical properties. Femora were applied to mechanical testing with a Microtest EM1/10/FR/m testing machine (Microtest, S.A., Madrid, Spain) and three-point bending strength was measured. Each bone was compressed with a constant load of 3 kN/s until failure. The load–displacement curve was obtained and converted into a stress–strain curve. Whole bone extrinsic biomechanical properties reported include ultimate load, ultimate displacement and work to failure. On the other hand, bone tissue intrinsic biomechanical properties include ultimate stress, ultimate strain and toughness. Mean values of immunosuppressants in blood, 24 h after administration, were: CsA (153± 20 ng/mL), FK-506 (4 ± 0.6 ng/mL) and RAPA (4 ±0.4 ng/mL). The group of rats treated with FK506 showed a significant decrease in ultimate load, ultimate displacement and work to failure with respect to all the studied groups and a decrease in ultimate strain and toughness against the rats treated with CsA and RAPA, without changes in ultimate stress. RAPA group showed a decrease in ultimate stress in respect to CONTROL group, and CsA group presented an increase in ultimate displacement, work to failure and ultimate strain versus CONTROL group. The above results agree with the previous findings about changes in BMD after immunosuppressants administration, showing that FK-506 is the immunosuppressive drug which produces the higher damage on bone tissue, being of minor importance that produced by RAPA. CsA did not affect negatively neither BMD nor biomechanical properties. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: M. Rubert: None Declared, D. Guede: None Declared, M. Montero: None Declared, J. R. Caeiro: None Declared, M. Martín-Fernández: None Declared, M. DiazCuriel: None Declared, C. De la Piedra Grant/Research Support from National Institute of Health FIS PI 06/0025. doi:10.1016/j.bone.2011.03.417
PP258-T Gender-specific modulation of bone remodelling by 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure and ablation of aryl hydrocarbon receptor alters material properties of bone matrix M.A.J. Finnilä a,⁎, M. Herlin b, P. Zioupos c, J. Risteli d, H.M. Miettinen e, M. Korkalainen e, T. Jämsä a, J. Tuukkanen f, H. Håkansson b, M. Viluksela e a Department of Medical Technology, University of Oulu, Oulu, Finland b Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden c Department of Engineering and Applied Sciences, Cranfield University, Shrivenham, UK d Department of Clinical Chemistry, University of Oulu, Oulu, Finland e Department of Environmental Health, National Institute for Health and Welfare, Kuopio, Finland f Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland Abstract: Bone is sensitive to dioxin exposure, which causes decrease in strength, cortical area and mineralization. Most of the toxic effects are suggested to be mediated through the aryl hydrocarbon reseptor (AhR), a regulator of cell metabolism, proliferation and differentiation. This study aims to further characterize the role of AhR in dioxin-induced bone toxicity using AhR knockout mice. Eight groups with six mice in each were studied. During the ten week experimentation a group of female and male Ahr+/+ and Ahr−/− mice were given either TCDD (total dose of 200 μg/kg) or corn oil vehicle (controls) by oral gavage. One week after the last dosing the mice were sacrificed. Left femurs were assessed for material properties by nanoindentation analysis. Twelve indentations with quasistatic loading protocol were done in two rows of six indents from endocortical to periosteal side at ventral side of transverse bone sections. Bone formation and resorption markers procollagen type I N-terminal propeptide (PINP) and Cterminal telopeptide of type I collagen (CTX), respectively, were measured from plasma. Females were more sensitive to both dioxin exposure and AhR ablation. TCDD exposure decreased PINP levels by 27% while AhR ablation increased PINP levels by 20%. Both elastic modulus and hardness showed reversed response to PINP levels. TCDD exposure increased hardness by 9% (p < 0.05) and elastic modulus 8% (p < 0.05). There was a slight 2.5% decrease in material properties in Ahr−/− groups, also opposite to increased PINP levels. TCDD also elevated CTX levels (33%) but not as much as AhR ablation (70%, p < 0.01). However, exposure to TCDD did not further increase CTX or PINP levels in Ahr−/− group. In males, neither TCDD exposure nor AhR ablation altered CTX or PINP levels, however, hardness was increased by 8% (p < 0.05) in male Ahr−/− group. Here we have shown that both TCDD and AhR can affect bone remodelling and material properties. Plasma PINP levels were inversely related to changes in material properties. Increased PINP lead to softer and elastic bone matrix seen in Ahr−/− group, while TCDD exposure decreases bone formation causing remaining tissue to be stiffer and harder, thus more senescent in nature. Increased bone resorption leads to higher cortical porosity resulting ultimately in weaker bones in TCDD exposed females as we have previously observed. This study further supports previous findings that females are more sensitive to the presence of AhR and TCDD exposure. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.418
PP259-S Femoral bone matrix mineralization is not altered in a rat model of type 2 diabetes mellitus N. Fratzl-Zelman a,⁎, P. Roschger a, S. Lueger a, K.-P. Guenther b, C. Goettsch b, L.C. Hofbauer b, K. Klaushofer a, C. Hamann b a Ludwig Boltzmann Institute of Osteology, Vienna, Austria b Dresden Technical University Medical Center, Dresden, Germany Abstract: The Zucker Diabetic Fatty (ZDF) rat is an established model of type 2 diabetes mellitus. The animals develop a diabetic phenotype after 8 weeks exhibiting obesity, a strong increase in serum glucose levels, and micro- and macrovascular complications. Reduced bone mineral density as well as delayed fracture repair was reported in the ZDF rats. This is consistent with ex vivo observations showing suppressed osteoblastogenesis and decreased mineral matrix formation (Haman, C et al. ECTS 2010). It is not clear at the moment, if at the material level, bone quality is also altered in the osteopenic diabetic animals. In the present study, we used quantitative backscattered electron imaging (qBEI) to assess bone mineralization density distribution (BMDD), in femurs of 21-week old diabetic rats. Proximal metaphyseal, epiphyseal and cortical midshaft bone of femurs were examined in ZDF (n = 6) and controls (n = 6) animals. Five BMDD parameters were evaluated: CaMean, the mean calcium content, CaPeak, the most frequent calcium content, CaWidth the heterogeneity of mineralization, CaLow, the fraction of lowly mineralized areas correspond-
Abstracts / Bone 48 (2011) S173–S186 ing to sites of primary mineralization and CaHigh, the fraction of high mineralized bone. CaHigh was only determined for metaphyseal bone. The BMDD curves exhibited a shift towards higher matrix mineralization in both diabetic and control samples from metaphyseal to epiphyseal and to cortical bone reflecting the differences in local average tissue age due to the different remodeling situations. However, the comparison of BMDDs from diabetic with control rats did not reveal any significant differences, except in the metaphyseal site, where CaWith (+25%, p = 0.041) and CaHigh (+ 53.5%, p = 0.013) were increased in ZDF rats. The results show that the mineralization pattern is in general not affected by the diabetic phenotype. The differences in CaWidth and CaHigh observed are due to an increased fraction of residual cartilage in the bone trabeculae, suggesting alterations in endochondral bone formation in the ZDF rats. Our findings indicate that mineralization density distribution is normal in this type 2 diabetes rat model and therefore other pathophysiological factors might be responsible for the observed impaired bone phenotype. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.419
PP260-M Association between bone stiffness and age. A micro-finite element analysis in women from the OFELY cohort N. Vilayphiou a,⁎, S. Boutroy a, E. Sornay-Rendu a, F. Munoz a, B. Van Rietbergen b, P. Delmas a, R. Chapurlat a a INSERM U831 and Université de Lyon, Lyon, France b Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands Abstract: Areal bone mineral density (aBMD) decreases with age in women, but our knowledge of bone biomechanical properties is limited, so we have studied the decline of bone strength with age, as assessed with micro-finite element analysis (μFEA) using high resolution peripheral QCT (HR-pQCT). So far, our cross-sectional analysis has involved 483 out of 841 women from the OFELY cohort who had an HR-pQCT radius scan at the 14th year of follow-up. There were 203 pre(preM) and 280 postmenopausal (postM) women, aged 40 ± 9 and 71 ± 9 years old respectively. In postM women, 101 women sustained fragility fractures during the follow-up. Distal radius HR-pQCT scans were used to measure volumetric BMD (vBMD) and microarchitecture parameters, after segmentation of cortical (Ct) bone with advanced algorithm (Burghardt, Nishiyama et al. Bone 2010). Estimated failure load was assessed by μFEA as the main outcome of radius bone strength, and the load to strength ratio Φ was derived to estimate the risk for wrist fracture. Total hip aBMD was recorded by DXA at the same visit. In preM women, most parameters did not vary with age. In postM women, Ct porosity and trabecular (Tb) heterogeneity increased importantly with age, respectively from 0.7 to 3.4% and from 210 to 580 μm in total (both p<0.001). Total, Ct and Tb vBMD decreased with age, as well as Ct thickness and Tb number (r=−0.39 to −0.54, p<0.001). The deterioration of microarchitecture with age resulted in a decrease of the μFE-failure load of −9 to −14% by decade, with an overall loss of a third of bone strength throughout the postmenopausal period compared to preM women. A total increase of Φ by 48% was observed, so that after the age of 70 more than half (70%) of the elderly women had a risk factor Φ>1. Considering Φ as a general factor of risk (not only wrist fracture), we compared the ROC curves of Φ and total hip aBMD, with respect to prevalent fractures. The area under the curves was slightly higher for Φ than total hip aBMD (AUC=0.80 and 0.77, respectively). However, sensitivity and specificity for fracture of the mechanical criterion Φ>1 were 73 and 74% respectively, whereas that of the T-score<−2.5 were 15 and 98% respectively. μFEA are still ongoing to complete this cross sectional dataset of the OFELY cohort, but those preliminary data show a consistent decline of bone strength with age at the radius. We suggest that the calculated load to strength ratio might be a relevant indicator of fracture risk. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: N. Vilayphiou Grant/Research Support from ECTS/Servier Fellowship 2009, S. Boutroy: None Declared, E. Sornay-Rendu: None Declared, F. MUNOZ: None Declared, B. Van Rietbergen Consulting fees from Scanco Medical AG, P. Delmas: None Declared, R. Chapurlat: None Declared. doi:10.1016/j.bone.2011.03.420
PP261-T The interplay between calcium homeostasis and bone mineralization — A computational approach P. Kollmannsberger a,⁎, C. Lukas a, P. Roschger b, P. Fratzl a, R. Weinkamer a a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany b Ludwig Boltzmann Institute of Osteology, 4th Department, Hanusch Hospital & UHK-Meidling, Vienna, Austria
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Abstract: The mineral flux into and out of bone due to remodeling and mineralizaton plays an important role in calcium homeostasis and is tightly controlled by hormones. Endocrine diseases result in disturbed plasma hormone and mineral levels and cause long-term changes in bone mineralization. These changes can be diagnosed by deviations of the heterogeneity of the mineral content within bone, as quantified by the bone mineralization density distribution (BMDD) from the healthy reference [1]. Despite the large amount of available data, an intuitive understanding of how plasma hormone levels affect bone mineralization is lacking due to the complexity of the bone-endocrine regulatory system. Computer simulations are therefore a valuable tool to quantify the role of different influencing factors and to interpret physiological data. Our investigations are based on measurements of the BMDD using quantitative backscattered electron imaging (qBEI), and plasma mineral and hormone data of the same patients. We present a theoretical model that quantitatively links mineral and hormone kinetics to the long-term evolution of the BMDD. The model integrates current knowledge about the interactions of calcium, parathyroid hormone (PTH), calcitriol and bone cells, and incorporates it into an established theoretical description of the BMDD as a function of bone remodeling and mineralization [2]. As an example of particular interest, we apply our model to experimental data from patients with mild primary hyperparathyroidism (PHPT) [3]. We show how the reported broadening of the BMDD peak and its shift to lower calcium content can be predicted quantitatively based on plasma mineral and hormone data. The results demonstrate that bone is itself part of a feedback loop that contributes to long-term calcium homeostasis. Our study highlights how computer simulations can help to characterize the role of different influencing factors and to study generic control principles underlying the bone-endocrine regulatory system. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. References [1] Roschger P, et al. Bone mineralization density distribution in health and disease. Bone 2008;42(3):456–66. [2] Ruffoni D, et al. The bone mineralization density distribution as a fingerprint of the mineralization process. Bone 2007;40(5):1308–19. [3] Roschger P, et al. New observations on bone quality in mild primary hyperparathyroidism as determined by quantitative backscattered electron imaging. J Bone Miner Res 2007;22 (5):717–23. doi:10.1016/j.bone.2011.03.421
PP262-S The effect of a disturbed mineralization process on the bone mineralization density distribution (BMDD) C. Lukas a, P. Kollmannsberger a, D. Ruffoni b, P. Roschger c, P. Fratzl a, R. Weinkamer a,⁎ a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany b Institute for Biomechanics, ETH Zurich, Zurich, Switzerland c Ludwig Boltzmann Institute of Osteology, 4th Department, Hanusch Hospital & UHK-Meidling, Vienna, Austria Abstract: Trabecular bone consists of a patchwork of bone packets with different mineral contents. This heterogeneity of bone can be measured, for instance, in the electron microscope using quantitative backscattered electron imaging (qBEI) and quantified in a frequency distribution called the bone mineralization density distribution (BMDD). The BMDD has proven to be a sensitive diagnostic tool for bone diseases [1]. Mathematical modeling allows connecting pathological changes in the BMDD with disturbances in bone remodeling and mineralization. The model was successfully applied to situations of changes in bone turnover occurring at the onset of menopause and following antiresorptive therapy [2]. One limitation of the comparison between theoretical prediction and BMDD measurements is due to the fact that the experimental data are affected by the stochastic nature of the backscattering of electrons and the finite acquisition time. We have now devised an approach using regularization tools to deconvolve and correct measured BMDDs. As a result, the reference BMDD for healthy human adults could be defined with improved precision. Moreover, the mathematical model was applied to situations where the mineralization process is disturbed. The inadequate mineralization in osteomalacia and the increased bone turnover at menopause both lead to a shift of the BMDD histogram towards lower mineral contents, in comparison to reference. With the use of mathematical modeling, it became possible to differentiate the time evolution of the BMDD, for both disease scenarios. The influence of Sr-treatment was also considered in the mathematical model, taking into account the difference in electron backscattering between Sr and Ca. When about 5% of the Ca atoms are replaced by Sr in the newly formed bone only [3], the model predicts a strong transient narrowing of the BMDD. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. References [1] Roschger P, et al. Bone mineralization density distribution in health and disease. Bone 2008;42(3):456–66.
Abstracts / Bone 48 (2011) S173–S186
properties in sheep. This study was performed on Polish Merino Sheep that were kept under standard rearing conditions to slaughter at the age of 5 months of life to obtain mandible and teeth for analyses. After initial analysis of weight, length, morphometric, densitometric and mechanical properties of the complete mandible, five teeth (I1-I3, C1 and PM2) were extracted and evaluated individually. Using quantitative computed tomography technique (Somatom Emotion apparatus, Siemens), volumetric bone mineral density (vBMD) and total bone volume (Bvol) of mandible was measured. 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 measurements of horizontal and vertical diameters of cross-section of mandible at 50% length of diastema. Using an INSTRON 3367 apparatus (Instron, USA) and three-point bending test, mechanical parameters such as maximum elastic strength (Wy) and ultimate strength (Wf) of the right and left mandible body were estimated. Furthermore, weight, length, total volume and mean vBMD of teeth was measured. The values of weight, length, Bvol, mean vBMD, vBMD of the cortical bone and calcium hydroxyapatite density of mandible reached 116±6 g, 219±3 mm, 72±cm3, 1.864±0.02 g/cm3, 2.451±0.02 g/cm3 and 1054±26 mgCaHA/mL, respectively. The values of A, Ix, MRWT, CI, Wy and Wf of mandible were 54±2 mm2, 265±21, 0.797±0.02, 44±1, 823±42 N and 1216±106 N, respectively. The values of weight, length, total volume and mean vBMD of teeth were within the ranges of 150±3 mg–783±35 mg, 19±0.3 mm–28±0.5 mm, 113±2 mm3–491± 21 mm3 and 2.947±0.038 g/cm3–3.054±0.034 g/cm3, respectively. In conclusion, this study showed that determination of densitometric, morphometric and mechanical properties of mandible and teeth may serve as a novel attractive model for further investigations on metabolic response of calcified tissues to physiological, pharmacological, nutritional and toxicological factors. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.416
PP257-M Effects of treatment with cyclosporine, FK-506 or rapamycin on femoral biomechanical properties in male adult healthy rats M. Rubert a,⁎, D. Guede b, M. Montero a, J.R. Caeiro c, M. Martín-Fernández a, M. Diaz-Curiel d, C. De la Piedra a a Biochemistry Investigation, Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz, Madrid, Spain b Trabeculae S.L., Technological Park of Galicia, Ourense, Spain c Traumatology and Orthopedic Surgery, University Hospital Complex of Santiago de Compostela, Santiago de Compostela, Spain d Internal Medicine, Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz, Madrid, Spain Abstract: One of the factors which can contribute to the severe bone loss after transplantation is the direct action of immunosuppressants on bone cells. In a previous work we found that tacrolimus (FK-506) administration produced a decrease in femoral (F) and lumbar (L) bone mineral density (BMD). Rapamycin (RAPA) only decreased FBMD and cyclosporine (CsA) did not affect L or FBMD in rats. The aim of this work was to study the effects produced by CsA, FK-506 and RAPA on femoral biomechanical properties in male adult healthy rats. Forty-eight (12/group), 5-month-old male Wistar rats were used. CsA (2 mg/kg/day), FK-506 (3 mg/kg/day), RAPA (1.25 mg/kg/day) or water (0.5 mL/rat/day, CONTROL group) were administered by oral gavage for 3 months. After sacrifice, blood levels of immunosuppressants were determined and the right femur was excised and cleaned for determination of biomechanical properties. Femora were applied to mechanical testing with a Microtest EM1/10/FR/m testing machine (Microtest, S.A., Madrid, Spain) and three-point bending strength was measured. Each bone was compressed with a constant load of 3 kN/s until failure. The load–displacement curve was obtained and converted into a stress–strain curve. Whole bone extrinsic biomechanical properties reported include ultimate load, ultimate displacement and work to failure. On the other hand, bone tissue intrinsic biomechanical properties include ultimate stress, ultimate strain and toughness. Mean values of immunosuppressants in blood, 24 h after administration, were: CsA (153± 20 ng/mL), FK-506 (4 ± 0.6 ng/mL) and RAPA (4 ±0.4 ng/mL). The group of rats treated with FK506 showed a significant decrease in ultimate load, ultimate displacement and work to failure with respect to all the studied groups and a decrease in ultimate strain and toughness against the rats treated with CsA and RAPA, without changes in ultimate stress. RAPA group showed a decrease in ultimate stress in respect to CONTROL group, and CsA group presented an increase in ultimate displacement, work to failure and ultimate strain versus CONTROL group. The above results agree with the previous findings about changes in BMD after immunosuppressants administration, showing that FK-506 is the immunosuppressive drug which produces the higher damage on bone tissue, being of minor importance that produced by RAPA. CsA did not affect negatively neither BMD nor biomechanical properties. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: M. Rubert: None Declared, D. Guede: None Declared, M. Montero: None Declared, J. R. Caeiro: None Declared, M. Martín-Fernández: None Declared, M. DiazCuriel: None Declared, C. De la Piedra Grant/Research Support from National Institute of Health FIS PI 06/0025. doi:10.1016/j.bone.2011.03.417
PP258-T Gender-specific modulation of bone remodelling by 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure and ablation of aryl hydrocarbon receptor alters material properties of bone matrix M.A.J. Finnilä a,⁎, M. Herlin b, P. Zioupos c, J. Risteli d, H.M. Miettinen e, M. Korkalainen e, T. Jämsä a, J. Tuukkanen f, H. Håkansson b, M. Viluksela e a Department of Medical Technology, University of Oulu, Oulu, Finland b Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden c Department of Engineering and Applied Sciences, Cranfield University, Shrivenham, UK d Department of Clinical Chemistry, University of Oulu, Oulu, Finland e Department of Environmental Health, National Institute for Health and Welfare, Kuopio, Finland f Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland Abstract: Bone is sensitive to dioxin exposure, which causes decrease in strength, cortical area and mineralization. Most of the toxic effects are suggested to be mediated through the aryl hydrocarbon reseptor (AhR), a regulator of cell metabolism, proliferation and differentiation. This study aims to further characterize the role of AhR in dioxin-induced bone toxicity using AhR knockout mice. Eight groups with six mice in each were studied. During the ten week experimentation a group of female and male Ahr+/+ and Ahr−/− mice were given either TCDD (total dose of 200 μg/kg) or corn oil vehicle (controls) by oral gavage. One week after the last dosing the mice were sacrificed. Left femurs were assessed for material properties by nanoindentation analysis. Twelve indentations with quasistatic loading protocol were done in two rows of six indents from endocortical to periosteal side at ventral side of transverse bone sections. Bone formation and resorption markers procollagen type I N-terminal propeptide (PINP) and Cterminal telopeptide of type I collagen (CTX), respectively, were measured from plasma. Females were more sensitive to both dioxin exposure and AhR ablation. TCDD exposure decreased PINP levels by 27% while AhR ablation increased PINP levels by 20%. Both elastic modulus and hardness showed reversed response to PINP levels. TCDD exposure increased hardness by 9% (p < 0.05) and elastic modulus 8% (p < 0.05). There was a slight 2.5% decrease in material properties in Ahr−/− groups, also opposite to increased PINP levels. TCDD also elevated CTX levels (33%) but not as much as AhR ablation (70%, p < 0.01). However, exposure to TCDD did not further increase CTX or PINP levels in Ahr−/− group. In males, neither TCDD exposure nor AhR ablation altered CTX or PINP levels, however, hardness was increased by 8% (p < 0.05) in male Ahr−/− group. Here we have shown that both TCDD and AhR can affect bone remodelling and material properties. Plasma PINP levels were inversely related to changes in material properties. Increased PINP lead to softer and elastic bone matrix seen in Ahr−/− group, while TCDD exposure decreases bone formation causing remaining tissue to be stiffer and harder, thus more senescent in nature. Increased bone resorption leads to higher cortical porosity resulting ultimately in weaker bones in TCDD exposed females as we have previously observed. This study further supports previous findings that females are more sensitive to the presence of AhR and TCDD exposure. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.418
PP259-S Femoral bone matrix mineralization is not altered in a rat model of type 2 diabetes mellitus N. Fratzl-Zelman a,⁎, P. Roschger a, S. Lueger a, K.-P. Guenther b, C. Goettsch b, L.C. Hofbauer b, K. Klaushofer a, C. Hamann b a Ludwig Boltzmann Institute of Osteology, Vienna, Austria b Dresden Technical University Medical Center, Dresden, Germany Abstract: The Zucker Diabetic Fatty (ZDF) rat is an established model of type 2 diabetes mellitus. The animals develop a diabetic phenotype after 8 weeks exhibiting obesity, a strong increase in serum glucose levels, and micro- and macrovascular complications. Reduced bone mineral density as well as delayed fracture repair was reported in the ZDF rats. This is consistent with ex vivo observations showing suppressed osteoblastogenesis and decreased mineral matrix formation (Haman, C et al. ECTS 2010). It is not clear at the moment, if at the material level, bone quality is also altered in the osteopenic diabetic animals. In the present study, we used quantitative backscattered electron imaging (qBEI) to assess bone mineralization density distribution (BMDD), in femurs of 21-week old diabetic rats. Proximal metaphyseal, epiphyseal and cortical midshaft bone of femurs were examined in ZDF (n = 6) and controls (n = 6) animals. Five BMDD parameters were evaluated: CaMean, the mean calcium content, CaPeak, the most frequent calcium content, CaWidth the heterogeneity of mineralization, CaLow, the fraction of lowly mineralized areas correspond-
Abstracts / Bone 48 (2011) S173–S186 ing to sites of primary mineralization and CaHigh, the fraction of high mineralized bone. CaHigh was only determined for metaphyseal bone. The BMDD curves exhibited a shift towards higher matrix mineralization in both diabetic and control samples from metaphyseal to epiphyseal and to cortical bone reflecting the differences in local average tissue age due to the different remodeling situations. However, the comparison of BMDDs from diabetic with control rats did not reveal any significant differences, except in the metaphyseal site, where CaWith (+25%, p = 0.041) and CaHigh (+ 53.5%, p = 0.013) were increased in ZDF rats. The results show that the mineralization pattern is in general not affected by the diabetic phenotype. The differences in CaWidth and CaHigh observed are due to an increased fraction of residual cartilage in the bone trabeculae, suggesting alterations in endochondral bone formation in the ZDF rats. Our findings indicate that mineralization density distribution is normal in this type 2 diabetes rat model and therefore other pathophysiological factors might be responsible for the observed impaired bone phenotype. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.419
PP260-M Association between bone stiffness and age. A micro-finite element analysis in women from the OFELY cohort N. Vilayphiou a,⁎, S. Boutroy a, E. Sornay-Rendu a, F. Munoz a, B. Van Rietbergen b, P. Delmas a, R. Chapurlat a a INSERM U831 and Université de Lyon, Lyon, France b Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands Abstract: Areal bone mineral density (aBMD) decreases with age in women, but our knowledge of bone biomechanical properties is limited, so we have studied the decline of bone strength with age, as assessed with micro-finite element analysis (μFEA) using high resolution peripheral QCT (HR-pQCT). So far, our cross-sectional analysis has involved 483 out of 841 women from the OFELY cohort who had an HR-pQCT radius scan at the 14th year of follow-up. There were 203 pre(preM) and 280 postmenopausal (postM) women, aged 40 ± 9 and 71 ± 9 years old respectively. In postM women, 101 women sustained fragility fractures during the follow-up. Distal radius HR-pQCT scans were used to measure volumetric BMD (vBMD) and microarchitecture parameters, after segmentation of cortical (Ct) bone with advanced algorithm (Burghardt, Nishiyama et al. Bone 2010). Estimated failure load was assessed by μFEA as the main outcome of radius bone strength, and the load to strength ratio Φ was derived to estimate the risk for wrist fracture. Total hip aBMD was recorded by DXA at the same visit. In preM women, most parameters did not vary with age. In postM women, Ct porosity and trabecular (Tb) heterogeneity increased importantly with age, respectively from 0.7 to 3.4% and from 210 to 580 μm in total (both p<0.001). Total, Ct and Tb vBMD decreased with age, as well as Ct thickness and Tb number (r=−0.39 to −0.54, p<0.001). The deterioration of microarchitecture with age resulted in a decrease of the μFE-failure load of −9 to −14% by decade, with an overall loss of a third of bone strength throughout the postmenopausal period compared to preM women. A total increase of Φ by 48% was observed, so that after the age of 70 more than half (70%) of the elderly women had a risk factor Φ>1. Considering Φ as a general factor of risk (not only wrist fracture), we compared the ROC curves of Φ and total hip aBMD, with respect to prevalent fractures. The area under the curves was slightly higher for Φ than total hip aBMD (AUC=0.80 and 0.77, respectively). However, sensitivity and specificity for fracture of the mechanical criterion Φ>1 were 73 and 74% respectively, whereas that of the T-score<−2.5 were 15 and 98% respectively. μFEA are still ongoing to complete this cross sectional dataset of the OFELY cohort, but those preliminary data show a consistent decline of bone strength with age at the radius. We suggest that the calculated load to strength ratio might be a relevant indicator of fracture risk. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: N. Vilayphiou Grant/Research Support from ECTS/Servier Fellowship 2009, S. Boutroy: None Declared, E. Sornay-Rendu: None Declared, F. MUNOZ: None Declared, B. Van Rietbergen Consulting fees from Scanco Medical AG, P. Delmas: None Declared, R. Chapurlat: None Declared. doi:10.1016/j.bone.2011.03.420
PP261-T The interplay between calcium homeostasis and bone mineralization — A computational approach P. Kollmannsberger a,⁎, C. Lukas a, P. Roschger b, P. Fratzl a, R. Weinkamer a a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany b Ludwig Boltzmann Institute of Osteology, 4th Department, Hanusch Hospital & UHK-Meidling, Vienna, Austria
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Abstract: The mineral flux into and out of bone due to remodeling and mineralizaton plays an important role in calcium homeostasis and is tightly controlled by hormones. Endocrine diseases result in disturbed plasma hormone and mineral levels and cause long-term changes in bone mineralization. These changes can be diagnosed by deviations of the heterogeneity of the mineral content within bone, as quantified by the bone mineralization density distribution (BMDD) from the healthy reference [1]. Despite the large amount of available data, an intuitive understanding of how plasma hormone levels affect bone mineralization is lacking due to the complexity of the bone-endocrine regulatory system. Computer simulations are therefore a valuable tool to quantify the role of different influencing factors and to interpret physiological data. Our investigations are based on measurements of the BMDD using quantitative backscattered electron imaging (qBEI), and plasma mineral and hormone data of the same patients. We present a theoretical model that quantitatively links mineral and hormone kinetics to the long-term evolution of the BMDD. The model integrates current knowledge about the interactions of calcium, parathyroid hormone (PTH), calcitriol and bone cells, and incorporates it into an established theoretical description of the BMDD as a function of bone remodeling and mineralization [2]. As an example of particular interest, we apply our model to experimental data from patients with mild primary hyperparathyroidism (PHPT) [3]. We show how the reported broadening of the BMDD peak and its shift to lower calcium content can be predicted quantitatively based on plasma mineral and hormone data. The results demonstrate that bone is itself part of a feedback loop that contributes to long-term calcium homeostasis. Our study highlights how computer simulations can help to characterize the role of different influencing factors and to study generic control principles underlying the bone-endocrine regulatory system. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. References [1] Roschger P, et al. Bone mineralization density distribution in health and disease. Bone 2008;42(3):456–66. [2] Ruffoni D, et al. The bone mineralization density distribution as a fingerprint of the mineralization process. Bone 2007;40(5):1308–19. [3] Roschger P, et al. New observations on bone quality in mild primary hyperparathyroidism as determined by quantitative backscattered electron imaging. J Bone Miner Res 2007;22 (5):717–23. doi:10.1016/j.bone.2011.03.421
PP262-S The effect of a disturbed mineralization process on the bone mineralization density distribution (BMDD) C. Lukas a, P. Kollmannsberger a, D. Ruffoni b, P. Roschger c, P. Fratzl a, R. Weinkamer a,⁎ a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany b Institute for Biomechanics, ETH Zurich, Zurich, Switzerland c Ludwig Boltzmann Institute of Osteology, 4th Department, Hanusch Hospital & UHK-Meidling, Vienna, Austria Abstract: Trabecular bone consists of a patchwork of bone packets with different mineral contents. This heterogeneity of bone can be measured, for instance, in the electron microscope using quantitative backscattered electron imaging (qBEI) and quantified in a frequency distribution called the bone mineralization density distribution (BMDD). The BMDD has proven to be a sensitive diagnostic tool for bone diseases [1]. Mathematical modeling allows connecting pathological changes in the BMDD with disturbances in bone remodeling and mineralization. The model was successfully applied to situations of changes in bone turnover occurring at the onset of menopause and following antiresorptive therapy [2]. One limitation of the comparison between theoretical prediction and BMDD measurements is due to the fact that the experimental data are affected by the stochastic nature of the backscattering of electrons and the finite acquisition time. We have now devised an approach using regularization tools to deconvolve and correct measured BMDDs. As a result, the reference BMDD for healthy human adults could be defined with improved precision. Moreover, the mathematical model was applied to situations where the mineralization process is disturbed. The inadequate mineralization in osteomalacia and the increased bone turnover at menopause both lead to a shift of the BMDD histogram towards lower mineral contents, in comparison to reference. With the use of mathematical modeling, it became possible to differentiate the time evolution of the BMDD, for both disease scenarios. The influence of Sr-treatment was also considered in the mathematical model, taking into account the difference in electron backscattering between Sr and Ca. When about 5% of the Ca atoms are replaced by Sr in the newly formed bone only [3], the model predicts a strong transient narrowing of the BMDD. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. References [1] Roschger P, et al. Bone mineralization density distribution in health and disease. Bone 2008;42(3):456–66.