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Time response of the canal network in cortical bone to sciatic neurectomy in growing rats: a synchrotron radiation μCT study
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Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
quantitative assessment of structure function relationships in tissue healing, growth and adaptation and are now also often used for precise phenotypic characterization of tissue response in tissue engineering, gene therapy and molecular biology. In conclusion, hierarchical bioimaging in combination with biocomputational approaches are well suited to investigate structure function relationships of natural and engineered biomaterials and can be used to improve our understanding of and to reduce failure in biomaterials and tissue engineering constructs. This will ultimately lead to better patient care and an increase quality of life.
T4.4 Imaging in Cellular and Molecular Biomechanics 7861 We, 14:00-14:30 (P35) In situ imaging o f bone cells: opportunities for elucidating a cellular basis of bone disease and challenges for clinical translation M.L. Knothe Tate 1,2. 1Department ef Biomedical and 2Department ef Mechanical & Aerospace Engineering Case Western Reserve University, Cleveland, OH, USA We are currently on the cusp of understanding the relationship between mechanical loading modulated transport and maintenance of cell viability in the relatively impermeable matrix of bone tissue [1]. Our previous work has demonstrated a microanatomical basis for bone diseases such as osteoporosis and disuse osteopenia. Cell viability has been shown to relate to the state of bone tissue in health and disease [2]. New developments in confocal imaging, micro-CT imaging and optical coherence tomography allow for increased understanding of bone cells in their natural milieu. In situ imaging provides a basis for understanding the correlation between osteocyte viability, the patency of the osteocyte syncytial network and the progression of osteoporosis. Once this foundation has been established, new insights may be exploited to reverse pathological effects of osteoporosis at early time points in the disease process. It is expected that the World Health Organization definition of osteoporosis (BMD decrease of at least 2.5 standard deviations below the mean) is associated with reduced cellular viability and integrity of the cellular syncytium, perhaps beyond the point of efficient self-repair. Our current research program strives to define a cellular measure of bone quality that will allow for earlier diagnosis and interventional therapy to slow or halt progression of the disease. Furthermore, an understanding of cellular indicators of osteoporosis may provide insight for development of prophylactic measures to prevent onset of the disease altogether. This talk aims to outline the opportunities for elucidating a cellular basis of bone disease using cutting edge in situ imaging modalities to understand the pathophysiology of bone cells in their native, bioactive milieu. These opportunities are assessed in light of challenges in translating these imaging technologies to the clinical arena. References [1] Knothe Tate ML. J Biomechanics 2003; 36: 1409-24. [2] Knothe Tate ML, et al. Advances in Osteoporotic Fracture Management 2: 9-14. 6525 We, 14:30-14:45 (P35) Time response o f the canal network in cortical bone to sciatic neurectomy in growing rats: a synchrotron radiation #CT study T. Matsumoto 1, M. Yoshino 1, K. Uesugi 2, M. Tanaka 1. 1Bioengineering Division, Osaka University Graduate School of Engineering Science, Toyonaka, Japan, 2SPring-8/Japan Synchrotron Radiation Research Institute, Kouto, Japan Using monochromatic synchrotron radiation ~tCT (SR~tCT), we evaluated the canal network structure and its dependence on sciatic neurectomy (NX) in growing rats. Tibiae were harvested from both hindlimbs of 9- and 14-weekold male Wistar rats (n =8, respectively) subjected to unilateral NX at 6 weeks old. Tibiae were also obtained from intact hindlimbs of another 6-week-old rats (n=5). Distal diaphyseal segments were imaged at 5.83-~tm resolution by SR~tCT using 20-keV X-rays (SPring-8). Reconstructed images were then translated into local mineral densities using a calibrated linear relationship between linear absorption coefficients and concentrations of K2HPO4 solution. Cortical canal network was segmented by simple thresholding at a bone mineral density of 0.82g/cm 3 and its structural properties were quantitated. In intact hindlimbs, the canal network showed biphasic changes with age, as represented by the increases followed by the decreases of canal volume fraction (Vof) and canal connectivity density (COD): Vof =2.1, 3.1, and 1.8% and CoD = 18, 41, and 21 mm -3 in 6-, 9-, and 14-week old rats, respectively (p < 0.05). NX depressed the initial development and accelerated the subsequent regression of canal network, showing 16% smaller Vof and 22% smaller CoD in 9-week old rats and 27% smaller Vof and 39% smaller CoD in 14-week old rats compared with those in the contralateral intact hindlimbs (p < 0.05). In addition, NX decreased canal density in transverse section by 12% in 14-week old rats (p <0.05). Bone mineral density (BMD) in intact hindlimbs was higher
Oral Presentations in 14-week old rats (1.36 vs. 1.30 and 1.32mg/cm 3 in 6- and 9-week old rats, respectively, p <0.05). NX increased BMD slightly but significantly by 0.8% in 9-week old rats, implying lower bone turnover in NX hindlimbs at this stage. These findings indicate that (1) cortical vascular structure shows a biphasic change during growth phase and (2) mechanical unloading reduces cortical vascularity, possibly being contributory to the reduction of bone perfusion and bone atrophy. 7843 We, 14:45-15:00 (P35) Cellular phenotyping o f the mouse skeleton using synchrotron based nano-computed t o m o g r a p h y P. Schneider 1, D. Webster 1, E. Wasserman 1, M. Stauber 1, M. Stampanoni 2, R. MiJller 1. 1Institute for Biomedical Engineering, University and ETH Zurich, Switzerland, 2Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland Age-related and osteoporotic bone fractures have a devastating effect on morbidity and mortality. Therefore, the study of bone mechanical properties is of vital importance. It was shown that bone strength is determined not only by bone mass, but also by other factors such as bone micro- and ultrastructure as well as genetic predisposition. With the improvement of synchrotron radiation based micro- and nanocomputed tomography (SR pCT and nanoCT), spatial resolutions down to a few hundred nanometer are now possible. This allowed for the first time to directly assess ultrastructural bone tissue properties, such as cell lacunae, vessel cavities as well as microcrack initiation and propagation. Our strategy is to use hierarchical bioimaging to identify, quantify and finally explain the factors contributing to bone strength and the response of bone to mechanical stimulation. Our studies in two genetically distinct inbred strains revealed that the number of osteocyte lacunae was different for both strains within cortical bone, highlighting a potential genetic contribution. Moreover, statistical analysis showed that lacuna volume and lacuna number scaled with bone size, whereas the mean volume of a single cell lacuna was unaffected by size and was rather constant but unique per strain. Finally, we observed that microcracks originate mostly in vessels and propagate along the osteocytic network. As a further step a recently developed in vivo compression device has been used to load murine caudal vertebrae in vivo in order to quantify load-induced anabolic activity in bone and eventually, to shed light on the genetic control of bone adaptation. In conclusion, we established a method to quantitatively assess trabecular and cortical bone down to a cellular level by using a true 3D SR nanoCT system. In the future we will relate the observed differences in ultrastructural bone morphometry to gene expression. This discussion has huge medical and social implications in terms of targeting and treating susceptible individuals who are subject to bone diseases like osteoporosis. 6766 We, 15:00-15:15 (P35) Calcium response in isolated chick osteocytes and osteoblasts to direct deformation M. Tanaka 1, ~ Aonuma 2, T. Adachi 1, H. Kamioka 3, T. Takano-Yamamoto 3, M. Hojo 1. 1Department of Mechanical Engineering and Science, Graduate School of Engineering, Kyoto University, Kyoto, Japan, 2Graduate School of Cultural Studies and Human Science, Kobe University, Kobe, Japan, 3Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan The osteocyte network system embedded in the bone matrix is considered as one of the important elements in the mechanosensory mechanism for the bone remodeling. Extensive in vivo reports have identified osteocytes as load-sensitive cells [1,2]. However, up-regulations of biological response in osteocytes are always accompanied by the biological response of osteoblasts. Therefore, it is significant to compare the real-time response to mechanical stress, in order to examine whether osteocytes are the primary mechanosensory cells in bone. In the present study, we observed the calcium response in isolated embryonic chick osteocytes and osteoblasts to the direct deformation by the glass micro needle to compare the short-time mechanosensory response of the osteocytes and osteoblasts. The changes of the intracellular Ca 2+ concentration, which is indicated by fluorescent dye: Fluo4-AM and Fura Red-AM, were observed by a confocal laser-scanning microscopy. The direct deformation was locally applied by touching by the needle. As a result, the responding cell rate to the direct deformation was about 16% in osteocytes. On the contrary, the responding cell rate in osteoblasts to the direct deformation was more than double of that in osteocytes. The change in the fluorescent ratio in osteoblasts was also significantly higher than that in osteocytes. In conclusion, the calcium response in isolated osteocytes showed less sensitive trend to the direct deformation than isolated osteoblasts.
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
quantitative assessment of structure function relationships in tissue healing, growth and adaptation and are now also often used for precise phenotypic characterization of tissue response in tissue engineering, gene therapy and molecular biology. In conclusion, hierarchical bioimaging in combination with biocomputational approaches are well suited to investigate structure function relationships of natural and engineered biomaterials and can be used to improve our understanding of and to reduce failure in biomaterials and tissue engineering constructs. This will ultimately lead to better patient care and an increase quality of life.
T4.4 Imaging in Cellular and Molecular Biomechanics 7861 We, 14:00-14:30 (P35) In situ imaging o f bone cells: opportunities for elucidating a cellular basis of bone disease and challenges for clinical translation M.L. Knothe Tate 1,2. 1Department ef Biomedical and 2Department ef Mechanical & Aerospace Engineering Case Western Reserve University, Cleveland, OH, USA We are currently on the cusp of understanding the relationship between mechanical loading modulated transport and maintenance of cell viability in the relatively impermeable matrix of bone tissue [1]. Our previous work has demonstrated a microanatomical basis for bone diseases such as osteoporosis and disuse osteopenia. Cell viability has been shown to relate to the state of bone tissue in health and disease [2]. New developments in confocal imaging, micro-CT imaging and optical coherence tomography allow for increased understanding of bone cells in their natural milieu. In situ imaging provides a basis for understanding the correlation between osteocyte viability, the patency of the osteocyte syncytial network and the progression of osteoporosis. Once this foundation has been established, new insights may be exploited to reverse pathological effects of osteoporosis at early time points in the disease process. It is expected that the World Health Organization definition of osteoporosis (BMD decrease of at least 2.5 standard deviations below the mean) is associated with reduced cellular viability and integrity of the cellular syncytium, perhaps beyond the point of efficient self-repair. Our current research program strives to define a cellular measure of bone quality that will allow for earlier diagnosis and interventional therapy to slow or halt progression of the disease. Furthermore, an understanding of cellular indicators of osteoporosis may provide insight for development of prophylactic measures to prevent onset of the disease altogether. This talk aims to outline the opportunities for elucidating a cellular basis of bone disease using cutting edge in situ imaging modalities to understand the pathophysiology of bone cells in their native, bioactive milieu. These opportunities are assessed in light of challenges in translating these imaging technologies to the clinical arena. References [1] Knothe Tate ML. J Biomechanics 2003; 36: 1409-24. [2] Knothe Tate ML, et al. Advances in Osteoporotic Fracture Management 2: 9-14. 6525 We, 14:30-14:45 (P35) Time response o f the canal network in cortical bone to sciatic neurectomy in growing rats: a synchrotron radiation #CT study T. Matsumoto 1, M. Yoshino 1, K. Uesugi 2, M. Tanaka 1. 1Bioengineering Division, Osaka University Graduate School of Engineering Science, Toyonaka, Japan, 2SPring-8/Japan Synchrotron Radiation Research Institute, Kouto, Japan Using monochromatic synchrotron radiation ~tCT (SR~tCT), we evaluated the canal network structure and its dependence on sciatic neurectomy (NX) in growing rats. Tibiae were harvested from both hindlimbs of 9- and 14-weekold male Wistar rats (n =8, respectively) subjected to unilateral NX at 6 weeks old. Tibiae were also obtained from intact hindlimbs of another 6-week-old rats (n=5). Distal diaphyseal segments were imaged at 5.83-~tm resolution by SR~tCT using 20-keV X-rays (SPring-8). Reconstructed images were then translated into local mineral densities using a calibrated linear relationship between linear absorption coefficients and concentrations of K2HPO4 solution. Cortical canal network was segmented by simple thresholding at a bone mineral density of 0.82g/cm 3 and its structural properties were quantitated. In intact hindlimbs, the canal network showed biphasic changes with age, as represented by the increases followed by the decreases of canal volume fraction (Vof) and canal connectivity density (COD): Vof =2.1, 3.1, and 1.8% and CoD = 18, 41, and 21 mm -3 in 6-, 9-, and 14-week old rats, respectively (p < 0.05). NX depressed the initial development and accelerated the subsequent regression of canal network, showing 16% smaller Vof and 22% smaller CoD in 9-week old rats and 27% smaller Vof and 39% smaller CoD in 14-week old rats compared with those in the contralateral intact hindlimbs (p < 0.05). In addition, NX decreased canal density in transverse section by 12% in 14-week old rats (p <0.05). Bone mineral density (BMD) in intact hindlimbs was higher
Oral Presentations in 14-week old rats (1.36 vs. 1.30 and 1.32mg/cm 3 in 6- and 9-week old rats, respectively, p <0.05). NX increased BMD slightly but significantly by 0.8% in 9-week old rats, implying lower bone turnover in NX hindlimbs at this stage. These findings indicate that (1) cortical vascular structure shows a biphasic change during growth phase and (2) mechanical unloading reduces cortical vascularity, possibly being contributory to the reduction of bone perfusion and bone atrophy. 7843 We, 14:45-15:00 (P35) Cellular phenotyping o f the mouse skeleton using synchrotron based nano-computed t o m o g r a p h y P. Schneider 1, D. Webster 1, E. Wasserman 1, M. Stauber 1, M. Stampanoni 2, R. MiJller 1. 1Institute for Biomedical Engineering, University and ETH Zurich, Switzerland, 2Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland Age-related and osteoporotic bone fractures have a devastating effect on morbidity and mortality. Therefore, the study of bone mechanical properties is of vital importance. It was shown that bone strength is determined not only by bone mass, but also by other factors such as bone micro- and ultrastructure as well as genetic predisposition. With the improvement of synchrotron radiation based micro- and nanocomputed tomography (SR pCT and nanoCT), spatial resolutions down to a few hundred nanometer are now possible. This allowed for the first time to directly assess ultrastructural bone tissue properties, such as cell lacunae, vessel cavities as well as microcrack initiation and propagation. Our strategy is to use hierarchical bioimaging to identify, quantify and finally explain the factors contributing to bone strength and the response of bone to mechanical stimulation. Our studies in two genetically distinct inbred strains revealed that the number of osteocyte lacunae was different for both strains within cortical bone, highlighting a potential genetic contribution. Moreover, statistical analysis showed that lacuna volume and lacuna number scaled with bone size, whereas the mean volume of a single cell lacuna was unaffected by size and was rather constant but unique per strain. Finally, we observed that microcracks originate mostly in vessels and propagate along the osteocytic network. As a further step a recently developed in vivo compression device has been used to load murine caudal vertebrae in vivo in order to quantify load-induced anabolic activity in bone and eventually, to shed light on the genetic control of bone adaptation. In conclusion, we established a method to quantitatively assess trabecular and cortical bone down to a cellular level by using a true 3D SR nanoCT system. In the future we will relate the observed differences in ultrastructural bone morphometry to gene expression. This discussion has huge medical and social implications in terms of targeting and treating susceptible individuals who are subject to bone diseases like osteoporosis. 6766 We, 15:00-15:15 (P35) Calcium response in isolated chick osteocytes and osteoblasts to direct deformation M. Tanaka 1, ~ Aonuma 2, T. Adachi 1, H. Kamioka 3, T. Takano-Yamamoto 3, M. Hojo 1. 1Department of Mechanical Engineering and Science, Graduate School of Engineering, Kyoto University, Kyoto, Japan, 2Graduate School of Cultural Studies and Human Science, Kobe University, Kobe, Japan, 3Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan The osteocyte network system embedded in the bone matrix is considered as one of the important elements in the mechanosensory mechanism for the bone remodeling. Extensive in vivo reports have identified osteocytes as load-sensitive cells [1,2]. However, up-regulations of biological response in osteocytes are always accompanied by the biological response of osteoblasts. Therefore, it is significant to compare the real-time response to mechanical stress, in order to examine whether osteocytes are the primary mechanosensory cells in bone. In the present study, we observed the calcium response in isolated embryonic chick osteocytes and osteoblasts to the direct deformation by the glass micro needle to compare the short-time mechanosensory response of the osteocytes and osteoblasts. The changes of the intracellular Ca 2+ concentration, which is indicated by fluorescent dye: Fluo4-AM and Fura Red-AM, were observed by a confocal laser-scanning microscopy. The direct deformation was locally applied by touching by the needle. As a result, the responding cell rate to the direct deformation was about 16% in osteocytes. On the contrary, the responding cell rate in osteoblasts to the direct deformation was more than double of that in osteocytes. The change in the fluorescent ratio in osteoblasts was also significantly higher than that in osteocytes. In conclusion, the calcium response in isolated osteocytes showed less sensitive trend to the direct deformation than isolated osteoblasts.