Colloids and Surfaces A: Physicochem. Eng.

Aspects 438 (2013) 199205

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Colloids and Surfaces A: Physicochemical and Engineering Aspects

journal homepage: www.elsevier.com/locate/colsurfa

Micro-computed tomography and compressive characterization of trabecular bone

A.Catarina Vale a,b , M.F.C. Pereira c , A. Maurcio c , P. Amaral a,d , L.G. Rosa a,d , A. Lopes b , A. Rodrigues b,e , J. Caetano-Lopes b , B. Vidal b , J. Monteiro e , J.E. Fonseca b,e , H. Canho b,e , M.Ftima Vaz a,d,
ICEMS, Av. Rovisco Pais, Lisbon, Portugal Rheumatology Research Unit, IMM, Faculdade de Medicina de Lisboa, Lisbon, Portugal c Centro de Petrologia e Geoqumica, IST, UTL, Av. Rovisco Pais, Lisbon, Portugal d Departamento de Engenharia Mecnica, IST, UTL, Av. Rovisco Pais, Lisbon, Portugal e Servic o de Reumatologia e Doenc as sseas Metablicas, HSM, Lisbon, Portugal
b a

h i g h l i g h t s
Trabecular bone is an open-cell foam made by an network of rods and plates. On two bone groups, the microstructural evaluation was performed with micro-CT. Compression tests were performed to evaluate the mechanical behaviour. Correlations between structural and mechanical parameters could be found. The Gibson and Ashbys models for cellular solids were applied to the two groups.

g r a p h i c a l

a b s t r a c t

a r t i c l e

i n f o

a b s t r a c t
Materials with a cellular structure are common in nature and an example of natural solid foam is trabecular bone. As for other materials, the microstructural features of cellular solids affect their mechanical response. The aim of this work is to study how the trabecular bone microarchitecture affects the mechanical properties of two types of bone and compare the results with models existing on the literature for solid cellular materials. In spite of several works which make a more medical analysis, this type of materials-science approach is rare. Two types of trabecular bone were studied, namely from male and female patients with osteoporotic (fragility) fractures and patients with coxarthrosis, that were submitted to total hip arthroplasty. Each sample was rst analyzed with micro-computed tomography, micro-CT, for structural assessment. Several parameters used to characterize the three-dimensional structure of trabecular bone were obtained, including the bone volume fraction. Cylinder samples were also tested under uniaxial compression and the Youngs modulus and the ultimate stress were determined. No statistical differences between the two bone groups in respect to the structural and mechanical properties were found. The structural parameters that correlate better with the mechanical properties are different for each bone assembly, being the trabecular separation on the coxarthrosis group and

Article history: Received 13 November 2012 Received in revised form 23 January 2013 Accepted 24 January 2013 Available online 14 February 2013 Keywords: Solid foams Trabecular bone Structural characterization Micro-computed tomography Compression

Corresponding author at: Departamento de Engenharia Mecnica, Instituto Superior Tcnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Tel.: +351 218417340. E-mail addresses: cat.cvale@gmail.com (A.Catarina Vale), fatima.vaz@ist.utl.pt (M.Ftima Vaz). 0927-7757/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.01.057

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the trabecular number on the osteoporotic group. However, both trabecular separation and trabecular number are strongly correlated with the bone volume fraction, i.e., to the bone relative density. The existing models of Gibson and Ashby were adapted to the relationship between stiffness and strength with the bone volume fraction. In both bone groups, the Youngs modulus is reduced with the decrease of the bone volume fraction following a quadratic law as happens in bending dominated open-cell foams. The ultimate strength is related to the bone volume fraction by a relationship with an exponent between 1 and 2, which indicates that during cell collapse, there is a mixture of two mechanisms: elastic buckling and brittle crushing. 2013 Elsevier B.V. All rights reserved.

1. Introduction
s

C2

BV TV

(4)

Trabecular bone is a three-dimensional solid foam, which exists at the epiphysis of long bones and within the vertebral body [1]. The trabecular bone has a cellular structure made of an interconnected network of rods and plates forming an open-cell foam [1,2]. Fractures on vertebrae, hip and wrist tend to start in the regions of trabecular bone that show a decreased bone mass and changes in the microarchitecture, which were probably affected by a disease mechanism [1,3]. The microstructural changes on trabecular bone may be measured using non-invasive techniques, in opposition to the mechanical properties. In this sense, a prediction of the mechanical properties through structural characterization is fundamental, for the evaluation of fracture risk and for the strategies of treatment. Although some correlations already exist, the relationship between structural parameters and mechanical properties is still an open question [46]. Micro-computed tomography (micro-CT) has becoming a leading technique on non-destructive/non-invasive structural evaluation and may be used to the visualization and quantication of the three-dimensional (3D) structure of bone. Micro-CT allows evaluating bone volume fraction, as well as, the architectural bone arrangement [79]. Different mechanical tests can be performed to evaluate the mechanical properties of trabecular bone. By far, the most common procedure is through compression tests, due to their simple procedure and to the fact that bones are often submitted to compression loads [7,1014]. The parameters obtained may be associated, for instance, with the density [1,2]. On solid foams, the Youngs modulus (E) and the compressive strength ( ) are related to the density ( ), through the models proposed by Gibson and Ashby [1,2]. These parameters are normalized to the values of a solid compact structure, respectively Es , s and s . On trabecular bone, the values of Es , s and s are, in general, taken, respectively, as 12 GPa, 136 MPa and 2000 kg/m3 [2]. The relative modulus E/Es and the relative strength / s are functions of the relative density / s which are given, respectively by Eqs. (1) and (2): E = C1 Es = C2
m

The relationship between structural and mechanical parameters, making use of the model of Gibson and Ashby (GA), is essentially performed for healthy trabecular bone [1,2]. Also most of the correlations found in the literature between the mechanical features and the micro-CT results are performed with healthy bone [8,9,16,17]. Studies which include the comparison of two groups of diseased bone are rare [4,18]. The trabecular bone characteristics may be affected by diseases as osteoporosis, which is responsible for the reduction in bone mass and for the microarchitectural deterioration of the tissue. Osteoarthritis, a pathology related to a breakdown of the lubrication process at joints, may also cause alterations on the trabecular bone properties. The aim of the present work was to correlate structural characteristics with mechanical properties of two groups of trabecular bone samples, and to identify the deformation mechanisms with the application of the Gibson and Ashby models. Although several studies exist on the structure and mechanical properties of bone, so far, none combines experimental results from two types of nonhealthy bone, with models proposed for cellular structures, given rise to a new materials approach, instead of a more clinical view.

2. Materials and methods 2.1. Materials The effect of two different pathologies on bone, osteoarthritis and osteoporosis, were evaluated, using samples of two groups of trabecular bone, respectively, from patients with a particular form of osteoarthritis, which is coxarthrosis (C) and from patients that underwent a fragility fracture (F), probably due to osteoporosis. Samples were obtained from male and female patients that were submitted to total hip arthroplasty at the Orthopaedic Department of Hospital de Santa Maria, Lisbon, Portugal. Thirty one patients, 11 with fractures (median age 81 years) and 20 with coxarthrosis (median age 71 years) were evaluated. After the surgical procedure, the femoral epiphyses were immediately stored at 80 C. Before testing, the material was defrosted. From each femoral head, two cylinders were extracted, in the highest in vivo loading direction, using perforating drills. The two samples, one for micro-CT and the other for compression, have diameters respectively of 5 and 15 mm (Fig. 1a) and approximately 15 and 30 mm in length. The cortical shell was removed in order to obtain only cylinders with cellular trabecular structure. Prior to compression tests, bone cylinders were de-fatted for 3 h using a solution of methanol and chloroform (1:1), and were hydrated overnight in phosphate buffered saline solution (PBS). The samples for micro-CT analysis were subjected to a more complex procedure which included ve steps: xation, dehydration, clearing, impregnation and inclusion with polymethylmethacrylate (PMMA) [19].

(1)
s n

(2)
s

where C1 and C2 are constants [1,2]. For a three dimensional cubic cell structure, C1 = 1, while C2 depends on the way how the cell collapses, being 0.3 if the cells deform by a brittle crushing mode [1]. The constants m and n may change, respectively, between 13 and 1.52 [1]. As the relative density / s is equivalent to the bone volume fraction, BV/TV, determined by micro-CT [15], Eqs. (1) and (2) may be written: E BV C1 Es TV
m

(3)

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Fig. 1. (a) Trabecular bone cylinder (diameter 15 mm); (b) Typical compression stressstrain curve that illustrates schematically the main parameters obtained: E, and WULT .

ULT

2.2. Micro-CT evaluation The micro-CT study was performed on a high resolution device, SkyScan 1172 (SkyScan, Kontich, Belgium). The micro-CT procedure involves three main steps: acquisition, reconstruction and image analysis. The acquisition was performed under the conditions of intensity 160 A, voltage of 60 kV and voxel size of 30 m. On the reconstruction phase, the projection images were reconstructed, in about 500 slices along the ZZ axis, with the software NRecon (SkyScan, Kontich, Belgium). The binarization was carried out, with a threshold value adjusted to the minimum of the global grayscale histogram from each specimen evaluated. On the third step, the reconstructed slice images were processed with the 3D image analysis software (CTAn and ANT software, SkyScan, Kontich, Belgium). The 3D rendering enabled the determination of the structural parameters for the analysis of trabecular bone structure [20,21]: Percent bone volume or Bone volume fraction (BV/TV, %), Bone specic surface (BS/BV, mm1 ), Structure model index (SMI, none), Trabecular thickness (Tb.Th, mm), Trabecular number (Tb.N, mm1 ), Trabecular separation (Tb.Sp, mm), Fractal dimension (FD, none), and Degree of anisotropy (DA, none). Notice that SMI reects the rod or plate nature of the bones structure, with SMI = 0 for plate-like and SMI = 3 for rod-like structures [16,21]. 2.3. Compression tests Uniaxial compression tests were performed in a universal testing machine (model 5566, Instron Corporation, Canton, USA), with a load cell of 10 kN and a cross-head speed of 0.1 mm/s. The testing machine was controlled with the software (Bluehill2, Instron Corporation, Canton, USA). Stressstrain curves were obtained from the load-displacement data which enable to assess the mechanical parameters: Youngs modulus, E (calculated as the slope of the stressstrain curve in the linear elastic region), ultimate stress, ULT (the maximum stress that the bone can support without failing), and energy to failure, WULT (measured by the area under the stressstrain curve until the ultimate stress). E, ULT and WULT evaluate, respectively the stiffness, strength and toughness. A typical stressstrain compression curve is exhibited in Fig. 1b), with an indication of the previously mentioned parameters.

2.4. Statistical analysis Statistical analysis was performed using the statistical software SAS (Institute Inc., Cary, NC, USA). ShapiroWilk test was used to assess the normality of the distributions. It was found that the parameters had non-normal distributions. Then, the MannWhitney (Wilcoxon) test was performed to assess comparisons between the two bone diseases groups. For each bone disease group, Spearmans correlation coefcients were obtained for correlation between each microarchitectural and the mechanical parameters. Differences were considered statistically signicant between groups for two-side p-value lower than 0.05, and for pvalue lower than 0.0001, it was considered that those differences was highly statistically signicant. 3. Results Fig. 2 shows micro-CT three-dimensional reconstructed images for groups C (coxarthrosis) and F (fragility fracture), both for male and female trabecular bone samples. Samples have different values of bone volume fraction, BV/TV. Table 1 presents the global results obtained from the trabecular microstructural analysis, where a comparative microstructural evaluation between two bone groups was made. The Mann Whitney (Wilcoxon) test revealed that no signicant difference

Table 1 Descriptive statistics of the micro-CT measurements for the two bone groups C (coxarthrosis) and F (fragility fracture). C (n = 20) Parameter Age (years) BV/TV (%) BS/BV (mm1 ) Tb.Th (mm) Tb.N (mm1 ) Tb.Sp (mm) SMI (none) DA (none) FD (none) Mean SD 70 14.165 19.405 0.217 0.621 0.770 1.777 0.942 2.078 5 7.553 5.261 0.047 0.244 0.127 0.504 0.043 0.129 Range 18 25.183 19.036 0.176 0.857 0.407 1.778 0.160 0.402 F (n = 11) Mean SD 81 13.813 18.947 0.210 0.648 0.754 1.723 0.914 2.106 5 5.543 2.853 0.020 0.227 0.099 0.326 0.079 0.080 Range 18 15.775 7.655 0.063 0.665 0.359 1.134 0.270 0.258

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In order to correlate the microstructural with the mechanical parameters, the Spearman correlation coefcients were determined for the coxarthrosis and the fragility fracture bone samples. Results are presented respectively in Tables 3 and 4. For example, the bone volume fraction correlates positively with Tb.N, Tb.Th and negatively with Tb.Sp. For C and F groups, the mechanical parameters, E, ULT and WULT exhibit positive coefcients, which mean they increase with an increase on BV/TV, Tb.N and FD. On the contrary, the same mechanical parameters decrease with an increase on Tb.Sp and SMI, also for both disease groups. When evaluating the correlation of E, ULT and WULT with the parameters BS/BV, Tb.Th and DA there are different Spearman coefcient signs, which mean they have different behaviors on coxarthrosis specimens and fragility fracture samples. While on the C group, the mechanical properties decrease with BS/BV and increase with Tb.Th, on the F group there is not a clear trend between the mechanical parameters and BS/BV and Tb.Th. The effect of the degree of anisotropy is not clear on the mechanical properties of the C bone samples, while on the F group the mechanical properties increase with DA. The higher values of the correlation coefcients between E, ULT and WULT and the structural parameters were obtained on the C group for the Tb.Sp, while for the F group, the higher values were given by the Tb.N. It means that the best microstructural predictor of the mechanical properties of the coxarthrosis group is the trabecular separation and for the fragility fracture group is the trabecular number. Both Tb.Sp of the C group and Tb.N of the F group are strongly correlated with BV/TV and, consequently with bone relative density. In order to make an analysis of the theoretical models, Eqs. (3) and (4) were represented together with the experimental results for relative stiffness and strength as a function of the bone volume fraction, for each bone group. Fig. 3(a) and (b) exhibits plots of E/Es versus BV/TV for C and F groups, respectively. On Fig. 3(c) and (d) the strength / s is graphically represented as a function of BV/TV, for both C and F groups. The straight lines of the plots correspond to different exponents of Eqs. (3) and (4), respectively m = 1; 2; 3 and n = 1; 15; 2, with C1 and C2 equal to 1. As mention in the literature that for bone, C1 may be different from the unit [22], this parameter was changed between 0.8 and 1. However, changes in the values of C1 did not provide many benets in the tting to the experimental results to both bone groups. Fig. 4 shows the experimental values of the relative strength, ULT / s versus BV/TV for (a) C group and (b) F group. Straight lines represent Eq. (4), with n = 15 and two values of C2 , 0.3 and 1. From Fig. 4, it can be seen that the classical prediction from the Gibson and Ashby model, i.e., C2 = 0.3, underestimates the experimental data. 4. Discussion An understanding of the trabecular bone properties is fundamental on engineering design of materials to replace or generate the bone tissue or on the evaluation of the fracture risk of hip femoral bone. Thus, the relationships between microstructural and mechanical properties are of extreme importance. In the present work, micro-CT evaluation revealed no signicant differences on the microstructural parameters, between diseases groups. This means that, for instance, the bone volume fraction is similar on both groups. Although other authors [4,18] describe microstructural differences between the two diseases, other authors mention that there are no distinctions between osteoporotic and osteoarthritic bone characteristics on early stages of this last pathology [23], which may be our case. Consistently with no structural variations, our study revealed that no signicant differences were found in the mechanical properties between groups C and F.

Fig. 2. Micro-CT three-dimensional rendered images from C group: (a) male (BV/TV = 18.35%) and (b) female (BV/TV = 16.61%) trabecular bone samples; images from F group samples: (c) male (BV/TV = 13.33%) and (d) female (BV/TV = 12.09%).

between diseases was found. On both groups, the mean value of BV/TV is very similar. The values obtained on the compressive tests are shown in Table 2. No signicant differences, also evaluated by the MannWhitney (Wilcoxon) test, were found in the mechanical parameters for both bone groups.

A.Catarina Vale et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 438 (2013) 199205 Table 2 Descriptive statistics of the compression tests for the two bone groups C (coxarthrosis) and F (fragility fracture). C (n = 20) Parameter E (MPa) ULT (MPa) WULT (N mm/mm3 ) Mean SD 443.281 154.286 9.045 4.226 0.171 0.152 Range 610.680 17.763 0.706 F (n = 11) Mean SD 502.449 224.347 10.722 4.673 0.219 0.141 Range

203

705.975 16.624 0.417

Table 3 Spearman correlation coefcients between trabecular microarchitecture and mechanical behaviour parameters for C group. BS/BV BV/TV (%) BS/BV (mm1 ) Tb.Th (mm) Tb.N (mm1 ) Tb.Sp (mm) SMI (none) DA (none) FD (none)
a b

Tb.Th 0.826b 0.948b

Tb.N 0.957b 0.814b 0.703a

Tb.Sp 0.866b 0.728b 0.637a 0.935b

SMI 0.909b 0.791b 0.673a 0.857b 0.742b

DA 0.379 0.400 0.428 0.358 0.431 0.425

FD 0.987b 0.890b 0.789b 0.964b 0.885b 0.932b 0.409

E 0.385 0.273 0.220 0.401 0.476a 0.379 0.075 0.404

ULT

WULT 0.257 0.273 0.117 0.217 0.283 0.280 0.051 0.285

0.913b

0.392 0.342 0.218 0.379 0.488a 0.404 0.070 0.412

p < 0.05. p < 0.0001.

The determination of the correlation coefcients between microstructural and mechanical parameters allowed obtaining some trends. There is a positive correlation of the bone volume fraction and trabecular number with the Youngs modulus and

the ultimate stress, on both F and C groups. A negative correlation between Tb.Sp and the parameters E and ULT is also found. These relationships are in accordance with the ndings of Nazarian et al. [8] for healthy bone. The best microstructural predictor

Fig. 3. Plots of E/Es versus BV/TV for (a) C group and (b) F group; Plots of different values of m and n.

ULT /

versus BV/TV for (c) C group and (d) F group. Straight lines represent Eqs. (3) and (4) with

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Table 4 Spearman correlation coefcients between trabecular microarchitecture and mechanical behaviour parameters for F group. BS/BV BV/TV (%) BS/BV (mm1 ) Tb.Th (mm) Tb.N (mm1 ) Tb.Sp (mm) SMI (none) DA (none) FD (none)
a b

Tb.Th
a

Tb.N
a

Tb.Sp
b

SMI
a

DA
b

FD 0.970 0.833a 0.663a 0.901b 0.672a 0.911b 0.208

b

E 0.526 0.348 0.017 0.654a 0.535 0.499 0.104 0.499

ULT

WULT 0.286 0.138 0.112 0.374 0.190 0.348 0.138 0.269

0.804

0.617 0.862a

0.940 0.663a 0.410

0.747 0.365 0.060 0.862a

0.871 0.719a 0.517 0.776a 0.599a

0.112 0.017 0.060 0.156 0.017 0.243

0.199 0.017 0.112 0.321 0.190 0.269 0.130 0.156

p < 0.05. p < 0.0001.

The experimental data of the Youngs modulus plotted against volume fraction, lies between the lines obtained with m = 1 and m = 3, either on F and C groups. This is in accordance with the results obtained for healthy bone, on which the Youngs modulus varied with the square of the density [1,2]. This exponent corresponds to a bending dominated mechanism for the elastic deformation of trabecular bone. There is evidence, from micro-CT analysis of healthy trabecular bone, that bending is the dominant mode of linear elastic deformation [9]. From the data obtained on the present work, it may be assumed that the mechanism of linear deformation is not affected by the disease. The scatter in the data is usually due to variations in trabecular architecture, namely cell shape and orientation [1,2]. The ultimate relative stress is plotted against volume fraction on Fig. 3(c) for the C group and on Fig. 3(d) for the F group. The compressive strength varies with volume fraction to a power between 1 and 2. In fact, several data points are closer to the line of slope 1.5, either on the coxarthrosis samples or on fragility fracture specimens. On the cellular solids models, n = 2 corresponds to failure by elastic buckling, while n = 1.5 is used for a mechanism of brittle crushing [1,2]. It may be reasonable to believe that both mechanisms of buckling and crushing are present in the yield deformation of trabecular bone, of both diseases. On healthy bone, failure is supposed to occur, only by elastic buckling [2,9]. 5. Conclusions In spite of several works, which make a more clinical analysis of the trabecular bone properties, the materialsscience approach to this type of cellular solid is not common, being complementary to the works presented in the literature. A structural and mechanical characterization of two groups of non-healthy bone, respectively with osteoarthritis and osteoporosis, was performed using micro-computed tomography and compressive tests. No clear distinctions were obtained on the comparison of the structural and mechanical parameters of the two groups. Statistical correlations allowed obtaining the best structural predictor of the mechanical properties, which was found to be different for each bone group. These two structural parameters, trabecular separation and trabecular number, are strongly correlated with the bone volume fraction, which is equivalent to the relative density. The Gibson and Ashby models, derived for open-cell foams, were applied to the two bone group samples, and all the experimental data fall into the boundaries dened by the models. It is worthy to mention, that there is a large scatter of the experimental data, which is common when using trabecular bone samples. The application of the models allowed concluding that in the linear elastic regime, non-healthy bone deforms by bending, in similitude to the healthy bone. However, on the collapse regime, the failure mechanism is different for healthy and non-healthy bone. Trabecular healthy bone collapses due to elastic buckling, while

Fig. 4. Plots ULT / s versus BV/TV for (a) C group and (b) F group. Straight lines represent Eq. (4) with different values of C2, for n = 1.5.

of the mechanical properties was found to be different for the two pathologies, being the trabecular separation on the C group and the trabecular number on the F group. However, both Tb.Sp and Tb.N are strongly correlated with BV/TV. In this sense, the mechanical properties are related to bone relative density. The variability of trabecular bone is high which means that the ability to predict properties is less good in comparison with articial foams. However, the GA models [1,2] allow to explain the mechanical properties trends with the density.

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the two groups of diseased bone deform by a mixture of the two mechanisms of elastic buckling and brittle crushing. Acknowledgements AC Vale thanks the Portuguese research foundation FCT (Fundac o para a Cincia e Tecnologia) for the Ph.D. fellowship grant SFRH/BD/48100/2008. MFC Pereira and A Maurcio acknowledge FEDER Funds through Programa Operacional Factores de Competitividade COMPETE, and FCT Project PEstOE/CTE/UI0098/2011. References
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