- Email: [email protected]
Functional mapping of bone on the micrometer-scale by scanning acoustic microscopy
Abstracts
PP197/NIOP03 Evidence for diminished skeletal load response in women who fracture early vs. those who fracture late in life C. Hamiltona,⁎, T.J. Beckb, A. Khaledc, S.A. Jamald, J.D. Adachie, J.P. Brownf, K.S. Davisonf The Canadian Multicentre Osteoporosis Study (CaMos) Research Group a Department of Exercise Sciences/Multidisciplinary Osteoporosis Research Program, University of Toronto/Women's College Hospital, Toronto, Canada b Quantum Medical Metrics LLC, Johns Hopkins University, Baltimore, 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 who 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 (b50 years, between 50 and 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.2012.02.386
PP198 Experimental quantification of Wolff's law in an in vivo loading model D. Ruffoni⁎, F.A. Schulte, F.M. Lambers, D. Webster, G. Kuhn, R. Müller Institute for Biomechanics, ETH Zurich, Zurich, Switzerland
S125
Abstract: The remarkable processes of adaption and repair in living bone are based on continuous resorption and formation of small bone packets. It is believed that bone remodeling is mechanically controlled; however, a quantitative description of the cell response to mechanical stimuli, also called Wolff's law, is still missing. Here, we characterized for the first time the mechanobiological rules controlling local bone formation and resorption. Our approach combined high resolution in vivo microcomputed tomography scans with in silico micro-finite element (micro-FE) analysis. We considered the sixth caudal vertebrae of C57BL/6 mice, subject to cyclic mechanical loading at a peak force of 8 N and at a frequency of 10 Hz three times a week for 4 weeks. Subsequent scans of the same animal at different time points were registered and, by comparing the two images, it was possible to distinguish between formed and resorbed voxels. The strain energy density (SED) at the bone surface was estimated with micro-FE by simulating the experimental loading conditions. The local mechanical stimuli were linked to the remodeling events by counting, for each value of SED at the bone surface, the relative fraction of voxels being formed and removed within two consecutive measurements. With this approach, we characterized Wolff's law in terms of probabilities of formation/resorption events to take place within a given time interval, knowing that the SED at the surface had a certain value. We found that the remodeling laws could be described by exponential functions (exponential rise for bone formation, R2 = 0.99, and exponential decay for bone resorption, R2 = 0.99). Three fitting parameters could be used to quantitatively describe the mechanosensory system in terms of (i) non-targeted remodeling, (ii) mechanical sensitivity (i.e., extent of variations in cell activity caused by the mechanical stimuli) and (iii) mechanical control (i.e., how fast cells vary the activity with increasing local mechanical stimuli). We found that mechanical loading decreased the amount of nontargeted resorption, increased the mechanical sensitivity and decreased the amount of mechanical control both for formation and resorption. It is hoped that such in vivo analysis will foster a better understanding and prediction of mechanobiological processes in the skeletal system. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.387
PP199 Functional mapping of bone on the micrometer-scale by scanning acoustic microscopy D. Fixa,⁎, S. Pucheggerb, C. Pilz-Allena, P. Roschgerc, S. Blouinc, P. Fratzla, R. Weinkamera a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany b Dept. Dynamics of Condensed Systems at the Faculty of Physics, University of Vienna, Austria c Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Med. Dept. Hanusch Hospital, Vienna, Austria Abstract: The bone material is highly heterogeneous on the sub-millimeter level due to the processes of bone remodeling and mineralization. The structural heterogeneity, e.g., of the mineral content of different bone packets, has important consequences on the functional heterogeneity. A quantitative position-resolved assessment of the mechanical properties of bone is necessary for a microscopic understanding of its crack propagation and fracture properties, and therefore the material quality. Scanning acoustic microscopy (SAM) gives the reflectivity of focused acoustic waves from the surface of the bone sample with a lateral resolution of 1–2 μm. The measured reflectivity depends on two characteristics of the bone sample, the stiffness and the mass density. The main advantages compared to mechanical characterization by nanoindentation are higher spatial resolution, non-destructiveness and measurement of the stiffness under native wet conditions. In our study cross-sections of human femoral bone were investigated by SAM. Three aspects are important in our approach: (i) propagation of both longitudinal and transversal acoustic waves was considered; (ii) acoustic waves of different wavelengths were used; and (iii) the SAM measurements were complemented by quantitative backscattered electron imaging (qBEI), which gives information about the mineral density and can be aligned to the reflectivity maps obtained with SAM. The combinations of these two methods allowed the following findings. Firstly, within regions of similar mineral content the acoustic reflectivity allows the distinction of single bone lamellae. This implies a different stiffness for alternating lamellae. Secondly, comparison of elasticity in regions with different density shows that higher density is not necessarily correlated with higher acoustic reflectivity. We can explain this observation as a consequence of the non-monotonous relation between reflectivity and stiffness. A characterization of bone samples using SAM in combination with qBEI provides a novel and time-efficient method to characterize bone material quality on the scale of individual bone packets. An evident clinical application is to complement structural
S126
Abstracts
images of bone biopsies (obtained, e.g., by electron microscopy) with functional images of the mechanical properties. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.388
PP200 Effect of specimen size on microCT measurements of cortical porosity F. Particelli⁎, M. Montesi, L. Mecozzi, D. De Pasquale, F. Baruffaldi Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy Abstract: Micro-CT imaging has become fundamental in morphometric investigation of bone. The accuracy of bone measurements can be influenced by a segmentation method that, through a threshold value, separates “bone” from “non bone” in the reconstructed grayscale datasets. In order to validate the characterization of human cortical bone microarchitecture using micro-CT, cortical porosity was compared with that obtained through histological examination [1,2]. Since studies often compare samples with a range of different sizes, the accurate measurement in specimens of unequal size is important. The aim of this work was to compare microtomographic and histological analyses of cortical bone biopsies of two different sizes, and to investigate how porosity is affected by errors when a different threshold value is applied. Sixteen cortical bone biopsies (8 with a diameter of 3 mm (group A) and 8 with a diameter of 1.5 mm (group B)) taken from the human femoral diaphysis were firstly included in PolyMethaMethylAcrylate (PMMA), examined by micro-CT and then the porosity was calculated through the dedicate software. Secondly, the cortical bone biopsies embedded in PMMA were sectioned to thin slices, observed at the microscope and then the porosity was determined through the microscope software. From each sample, the cross section image obtained by micro-CT and the corresponding slice obtained by histology were compared, and different threshold values for the two groups were found. The porosities calculated by the two techniques were highly correlated (R2 = 0.93 for 3 mm diameter and R2 = 0.98 for 1.5 mm diameter). No statistically significant differences were shown between micro-CT and histology. Moreover, calculating porosity of group B using the threshold value obtained for the group A the difference d and the mean percentage difference d% found in the comparison are indicated in Table 1. Although with the little number of samples, this preliminary study confirmed that micro-CT analyses is a reliable method for the morphometrical characterization of cortical tissues. Moreover, these results demonstrate that, halving the diameters of cortical bone biopsies, a little difference in threshold value introduce a very small error in the porosity measurements. Table 1 d
SD
MicroCT threshold group A—threshold group B Porosity (%) 0.87 0.13
d%
SD
13.61
4.0
This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. References [1] Particelli et al. 2011, J Microsc. doi: 10.1111/j.1365-2818.2011.03573.x. [Epub ahead of print]. [2] Perilli E. et al. 2007, J Microsc 225:192-20. doi:10.1016/j.bone.2012.02.389
PP201 External mechanical microstimuli improve osseointegration of titanium implants in rat proximal tibiae G. Zacchettia,⁎, A. Wiskottb, J. Cugnonic, I. Botsisc, M. Piccininic, P. Ammanna a Division of Bone Disease, Dep. of Rehabilitation and Geriatrics, Switzerland b Laboratory of biomaterials, School of Dentistry, University of Geneva, Geneva, Switzerland c Laboratory of Applied Mechanics and Reliability Analysis, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Abstract: A poor osseointegration of endosseous implants is the cause of early implant failure compromising the outcome of a surgical intervention both in dentistry and orthopedics. The aim of this work was to measure the effect of external mechanical microstimuli of controlled magnitude on implant osseointegration in rat proximal tibiae. Increasing loads were selected and the dose-dependent effect on parameters of implant osseointegration was examined. 40 female rats 6 months old were operated at the right tibiae by transcutaneous insertion of two stepped titanium cylindrical implants, 1 mm and 0.8 mm in diameter, respectively. The loaded implant (LI) was fixed into the trabecular bone of the secondary spongiosa whereas the anchorage implant was inserted 8 mm distally, encompassing both cortical surfaces. After 2 weeks rats were assigned to 4 groups (Non Stimulated= NS; 1N, 2N 3N), and further underwent a daily external mechanical loading during 4 weeks. Parameters of bone architecture were analyzed ex-vivo by microcomputerized tomography (μCT) on a circular band of 0.5 mm around the LI as well as in a region located up to 1 mm away from the LI, along the axis of the force. Ultimate Strength (US) was measured by a pull-out test as indicator of implant osseointegration. Increasing loading did not significantly modify BV/TV around the LI; still trabecular BV/TV was increased in the vicinity of the LI in 1N rats and decreased in 3N rats, while Cortical Thickness (Ct.Th) significantly increased in the 3N group. 2N rats displayed a significant increase of the pull-out force necessary to loosen the implant compared to NS, corresponding to the load having a partial effect on trabecular and cortical bone near LI. We hypothesize that bone ingrowths within the etched implant surface may improve implant anchorage. In conclusion, the in-vivo mechanical loading of implants induces loaddependent modifications in bone microarchitecture and improves implant osseointegration in rat tibiae. The histomorphometric analysis of bone growing on the implant surface is necessary to further explain the mechanism of implant osseointegration.
BV/TV Ct.Th (mm) US (N)
NS
1N (750 με)
2N (1500 με)
3N (2250 με)
0.15 ± 0.02 0.44 ± 0.01 39.57 ± 2.23
0.20 ± 0.02 0.43 ± 0.01 40.82 ± 3.12
0.17 ± 0.02 0.43 ± 0.01 46.63 ± 2.21*
0.14 ± 0.02# 0.46 ± 0.01# 43.81 ± 3.41
Average ± SEM; #p b 0.05 vs 1N; *p b 0.05 vs NS. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.390
PP202 Effects of PEMF and sulfurous mineral water on bone in experimental osteoporosis G. Stefanovskia,⁎, T. Popovića, M. Stefanovskib a Rheumatology-osteoporosis, Institute for Physical Medicine and Rehabilitation, Banja Luka, Bosnia and Herzegovina b Balneology, Institut for Balnelogy, PMR Mlječanica, Koz. Dubica, Bosnia and Herzegovina Abstract: Introduction: Sulfurous mineral water is used in the treatment of skeletal disorders. Electric and magnetic fields could modify the behavior of bone cells. Aim of study: The aim of our study was to evaluate the influence of PEMF and sulfurous mineral water “Mljecanica” on bone in a model of estrogen-deficient osteoporosis. Material and methods: Fourteen-weeks-old bilaterally ovariectomized female Wistar rats (n = 21) were randomly assigned to three groups: OVX + PEMF + SW (n = 7), OVW + SW (n = 7) and OVX control (n = 7). The accommodation condition (temperature 22°–24 °C) and feeding were the same for all groups. After six weeks OVX+PEMF + SW rats were supplied with sulfurous mineral water ad libitum and exposed to PEMF Kosmag 60 (40 Hz, 10 mT, 45 min) during four weeks, 5 days per week. OVX + SW rats were supplied with sulfurous water ad libitum, during four weeks, 5 days per week. OVX animals were drinking taped water ad libitum. After four weeks animals were sacrificed. At the end of the four-week period biochemical analyses: osteocalcin (OC), alkaline phosphatase (AP), calcium (Ca) and phosphorus (P) were evaluated. The histological analyses of left tibia were studied by routine light microscope. Biomechanical properties were studied on TOMI 2001. Results: Statistically significant increase of OC (p b 0.05) and statistically significant decrease of Ca and AP (p b 0.01) were obtained in OVX+PEMF + SW compared to OVX-control group. The increase of same parameters in OVX + SW group was not significant in comparison with that of OVX group. Histological observation in OVX+PEMF + SW showed growth of young chondrocytes in the central zone and cartilage on peripheral parts. The trabecules were thicker, still disconnected, with bone marrow in between. OVX + SW showed the growth of young chondrocytes in the central zone of bone and their migration to peripheral parts. Biomechanical analyses of the left femur on banding and torsion in experimental groups showed better quality of bone. Conclusions: This study shows that sulfurous water, used in experimental osteoporosis, induces the reparing mechanism of osteoporotic bone. In group OVX+ PEMF + SW we observed synergic effect on calcium level and positive impacts on the mechanical properties of bone. Treatment with PEMF and sulfurous water decreased bone fragility.
PP197/NIOP03 Evidence for diminished skeletal load response in women who fracture early vs. those who fracture late in life C. Hamiltona,⁎, T.J. Beckb, A. Khaledc, S.A. Jamald, J.D. Adachie, J.P. Brownf, K.S. Davisonf The Canadian Multicentre Osteoporosis Study (CaMos) Research Group a Department of Exercise Sciences/Multidisciplinary Osteoporosis Research Program, University of Toronto/Women's College Hospital, Toronto, Canada b Quantum Medical Metrics LLC, Johns Hopkins University, Baltimore, 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 who 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 (b50 years, between 50 and 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.2012.02.386
PP198 Experimental quantification of Wolff's law in an in vivo loading model D. Ruffoni⁎, F.A. Schulte, F.M. Lambers, D. Webster, G. Kuhn, R. Müller Institute for Biomechanics, ETH Zurich, Zurich, Switzerland
S125
Abstract: The remarkable processes of adaption and repair in living bone are based on continuous resorption and formation of small bone packets. It is believed that bone remodeling is mechanically controlled; however, a quantitative description of the cell response to mechanical stimuli, also called Wolff's law, is still missing. Here, we characterized for the first time the mechanobiological rules controlling local bone formation and resorption. Our approach combined high resolution in vivo microcomputed tomography scans with in silico micro-finite element (micro-FE) analysis. We considered the sixth caudal vertebrae of C57BL/6 mice, subject to cyclic mechanical loading at a peak force of 8 N and at a frequency of 10 Hz three times a week for 4 weeks. Subsequent scans of the same animal at different time points were registered and, by comparing the two images, it was possible to distinguish between formed and resorbed voxels. The strain energy density (SED) at the bone surface was estimated with micro-FE by simulating the experimental loading conditions. The local mechanical stimuli were linked to the remodeling events by counting, for each value of SED at the bone surface, the relative fraction of voxels being formed and removed within two consecutive measurements. With this approach, we characterized Wolff's law in terms of probabilities of formation/resorption events to take place within a given time interval, knowing that the SED at the surface had a certain value. We found that the remodeling laws could be described by exponential functions (exponential rise for bone formation, R2 = 0.99, and exponential decay for bone resorption, R2 = 0.99). Three fitting parameters could be used to quantitatively describe the mechanosensory system in terms of (i) non-targeted remodeling, (ii) mechanical sensitivity (i.e., extent of variations in cell activity caused by the mechanical stimuli) and (iii) mechanical control (i.e., how fast cells vary the activity with increasing local mechanical stimuli). We found that mechanical loading decreased the amount of nontargeted resorption, increased the mechanical sensitivity and decreased the amount of mechanical control both for formation and resorption. It is hoped that such in vivo analysis will foster a better understanding and prediction of mechanobiological processes in the skeletal system. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.387
PP199 Functional mapping of bone on the micrometer-scale by scanning acoustic microscopy D. Fixa,⁎, S. Pucheggerb, C. Pilz-Allena, P. Roschgerc, S. Blouinc, P. Fratzla, R. Weinkamera a Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany b Dept. Dynamics of Condensed Systems at the Faculty of Physics, University of Vienna, Austria c Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 4th Med. Dept. Hanusch Hospital, Vienna, Austria Abstract: The bone material is highly heterogeneous on the sub-millimeter level due to the processes of bone remodeling and mineralization. The structural heterogeneity, e.g., of the mineral content of different bone packets, has important consequences on the functional heterogeneity. A quantitative position-resolved assessment of the mechanical properties of bone is necessary for a microscopic understanding of its crack propagation and fracture properties, and therefore the material quality. Scanning acoustic microscopy (SAM) gives the reflectivity of focused acoustic waves from the surface of the bone sample with a lateral resolution of 1–2 μm. The measured reflectivity depends on two characteristics of the bone sample, the stiffness and the mass density. The main advantages compared to mechanical characterization by nanoindentation are higher spatial resolution, non-destructiveness and measurement of the stiffness under native wet conditions. In our study cross-sections of human femoral bone were investigated by SAM. Three aspects are important in our approach: (i) propagation of both longitudinal and transversal acoustic waves was considered; (ii) acoustic waves of different wavelengths were used; and (iii) the SAM measurements were complemented by quantitative backscattered electron imaging (qBEI), which gives information about the mineral density and can be aligned to the reflectivity maps obtained with SAM. The combinations of these two methods allowed the following findings. Firstly, within regions of similar mineral content the acoustic reflectivity allows the distinction of single bone lamellae. This implies a different stiffness for alternating lamellae. Secondly, comparison of elasticity in regions with different density shows that higher density is not necessarily correlated with higher acoustic reflectivity. We can explain this observation as a consequence of the non-monotonous relation between reflectivity and stiffness. A characterization of bone samples using SAM in combination with qBEI provides a novel and time-efficient method to characterize bone material quality on the scale of individual bone packets. An evident clinical application is to complement structural
S126
Abstracts
images of bone biopsies (obtained, e.g., by electron microscopy) with functional images of the mechanical properties. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.388
PP200 Effect of specimen size on microCT measurements of cortical porosity F. Particelli⁎, M. Montesi, L. Mecozzi, D. De Pasquale, F. Baruffaldi Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy Abstract: Micro-CT imaging has become fundamental in morphometric investigation of bone. The accuracy of bone measurements can be influenced by a segmentation method that, through a threshold value, separates “bone” from “non bone” in the reconstructed grayscale datasets. In order to validate the characterization of human cortical bone microarchitecture using micro-CT, cortical porosity was compared with that obtained through histological examination [1,2]. Since studies often compare samples with a range of different sizes, the accurate measurement in specimens of unequal size is important. The aim of this work was to compare microtomographic and histological analyses of cortical bone biopsies of two different sizes, and to investigate how porosity is affected by errors when a different threshold value is applied. Sixteen cortical bone biopsies (8 with a diameter of 3 mm (group A) and 8 with a diameter of 1.5 mm (group B)) taken from the human femoral diaphysis were firstly included in PolyMethaMethylAcrylate (PMMA), examined by micro-CT and then the porosity was calculated through the dedicate software. Secondly, the cortical bone biopsies embedded in PMMA were sectioned to thin slices, observed at the microscope and then the porosity was determined through the microscope software. From each sample, the cross section image obtained by micro-CT and the corresponding slice obtained by histology were compared, and different threshold values for the two groups were found. The porosities calculated by the two techniques were highly correlated (R2 = 0.93 for 3 mm diameter and R2 = 0.98 for 1.5 mm diameter). No statistically significant differences were shown between micro-CT and histology. Moreover, calculating porosity of group B using the threshold value obtained for the group A the difference d and the mean percentage difference d% found in the comparison are indicated in Table 1. Although with the little number of samples, this preliminary study confirmed that micro-CT analyses is a reliable method for the morphometrical characterization of cortical tissues. Moreover, these results demonstrate that, halving the diameters of cortical bone biopsies, a little difference in threshold value introduce a very small error in the porosity measurements. Table 1 d
SD
MicroCT threshold group A—threshold group B Porosity (%) 0.87 0.13
d%
SD
13.61
4.0
This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. References [1] Particelli et al. 2011, J Microsc. doi: 10.1111/j.1365-2818.2011.03573.x. [Epub ahead of print]. [2] Perilli E. et al. 2007, J Microsc 225:192-20. doi:10.1016/j.bone.2012.02.389
PP201 External mechanical microstimuli improve osseointegration of titanium implants in rat proximal tibiae G. Zacchettia,⁎, A. Wiskottb, J. Cugnonic, I. Botsisc, M. Piccininic, P. Ammanna a Division of Bone Disease, Dep. of Rehabilitation and Geriatrics, Switzerland b Laboratory of biomaterials, School of Dentistry, University of Geneva, Geneva, Switzerland c Laboratory of Applied Mechanics and Reliability Analysis, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Abstract: A poor osseointegration of endosseous implants is the cause of early implant failure compromising the outcome of a surgical intervention both in dentistry and orthopedics. The aim of this work was to measure the effect of external mechanical microstimuli of controlled magnitude on implant osseointegration in rat proximal tibiae. Increasing loads were selected and the dose-dependent effect on parameters of implant osseointegration was examined. 40 female rats 6 months old were operated at the right tibiae by transcutaneous insertion of two stepped titanium cylindrical implants, 1 mm and 0.8 mm in diameter, respectively. The loaded implant (LI) was fixed into the trabecular bone of the secondary spongiosa whereas the anchorage implant was inserted 8 mm distally, encompassing both cortical surfaces. After 2 weeks rats were assigned to 4 groups (Non Stimulated= NS; 1N, 2N 3N), and further underwent a daily external mechanical loading during 4 weeks. Parameters of bone architecture were analyzed ex-vivo by microcomputerized tomography (μCT) on a circular band of 0.5 mm around the LI as well as in a region located up to 1 mm away from the LI, along the axis of the force. Ultimate Strength (US) was measured by a pull-out test as indicator of implant osseointegration. Increasing loading did not significantly modify BV/TV around the LI; still trabecular BV/TV was increased in the vicinity of the LI in 1N rats and decreased in 3N rats, while Cortical Thickness (Ct.Th) significantly increased in the 3N group. 2N rats displayed a significant increase of the pull-out force necessary to loosen the implant compared to NS, corresponding to the load having a partial effect on trabecular and cortical bone near LI. We hypothesize that bone ingrowths within the etched implant surface may improve implant anchorage. In conclusion, the in-vivo mechanical loading of implants induces loaddependent modifications in bone microarchitecture and improves implant osseointegration in rat tibiae. The histomorphometric analysis of bone growing on the implant surface is necessary to further explain the mechanism of implant osseointegration.
BV/TV Ct.Th (mm) US (N)
NS
1N (750 με)
2N (1500 με)
3N (2250 με)
0.15 ± 0.02 0.44 ± 0.01 39.57 ± 2.23
0.20 ± 0.02 0.43 ± 0.01 40.82 ± 3.12
0.17 ± 0.02 0.43 ± 0.01 46.63 ± 2.21*
0.14 ± 0.02# 0.46 ± 0.01# 43.81 ± 3.41
Average ± SEM; #p b 0.05 vs 1N; *p b 0.05 vs NS. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.390
PP202 Effects of PEMF and sulfurous mineral water on bone in experimental osteoporosis G. Stefanovskia,⁎, T. Popovića, M. Stefanovskib a Rheumatology-osteoporosis, Institute for Physical Medicine and Rehabilitation, Banja Luka, Bosnia and Herzegovina b Balneology, Institut for Balnelogy, PMR Mlječanica, Koz. Dubica, Bosnia and Herzegovina Abstract: Introduction: Sulfurous mineral water is used in the treatment of skeletal disorders. Electric and magnetic fields could modify the behavior of bone cells. Aim of study: The aim of our study was to evaluate the influence of PEMF and sulfurous mineral water “Mljecanica” on bone in a model of estrogen-deficient osteoporosis. Material and methods: Fourteen-weeks-old bilaterally ovariectomized female Wistar rats (n = 21) were randomly assigned to three groups: OVX + PEMF + SW (n = 7), OVW + SW (n = 7) and OVX control (n = 7). The accommodation condition (temperature 22°–24 °C) and feeding were the same for all groups. After six weeks OVX+PEMF + SW rats were supplied with sulfurous mineral water ad libitum and exposed to PEMF Kosmag 60 (40 Hz, 10 mT, 45 min) during four weeks, 5 days per week. OVX + SW rats were supplied with sulfurous water ad libitum, during four weeks, 5 days per week. OVX animals were drinking taped water ad libitum. After four weeks animals were sacrificed. At the end of the four-week period biochemical analyses: osteocalcin (OC), alkaline phosphatase (AP), calcium (Ca) and phosphorus (P) were evaluated. The histological analyses of left tibia were studied by routine light microscope. Biomechanical properties were studied on TOMI 2001. Results: Statistically significant increase of OC (p b 0.05) and statistically significant decrease of Ca and AP (p b 0.01) were obtained in OVX+PEMF + SW compared to OVX-control group. The increase of same parameters in OVX + SW group was not significant in comparison with that of OVX group. Histological observation in OVX+PEMF + SW showed growth of young chondrocytes in the central zone and cartilage on peripheral parts. The trabecules were thicker, still disconnected, with bone marrow in between. OVX + SW showed the growth of young chondrocytes in the central zone of bone and their migration to peripheral parts. Biomechanical analyses of the left femur on banding and torsion in experimental groups showed better quality of bone. Conclusions: This study shows that sulfurous water, used in experimental osteoporosis, induces the reparing mechanism of osteoporotic bone. In group OVX+ PEMF + SW we observed synergic effect on calcium level and positive impacts on the mechanical properties of bone. Treatment with PEMF and sulfurous water decreased bone fragility.