Three-dimensional characterization of cortical bone microstructure by microcomputed tomography: validation with ultrasonic and microscopic measurements.

BACKGROUND: The porosity of human cortical bone is one of the major parameters conditioning bone strength. The purpose of this study was to validate the characterization of human cortical bone microarchitecture using microcomputed tomography (microCT). To validate this microCT technique, the structural measurements were compared with other methods such as ultrasonic techniques and scanning electron microscopy (SEM). METHODS: Nineteen cortical samples were extracted from the superior, middle, and inferior shaft of three human femurs (FI, FII, FIII). The samples were scanned by microCT with an isotropic resolution of 8 microm. Most of the structural parameters used for trabecular microarchitecture were calculated to characterize the network of pores. On the same cortical samples, (1) ultrasound measurements were performed using contact transmission emitter-receptor to determine elastic coefficient and Young's modulus; (2) SEM was performed on femoral cross sections from FII to evaluate the porosity. RESULTS: The morphological parameters showed a wide range of variation depending of the level of the diaphysis. Porosity measured by microCT was significantly correlated with porosity measured by SEM (r = 0.91, P < 0.05). Moreover, all the morphological parameters showed high correlation coefficients with the elastic coefficient and Young's modulus, leading to validation of our three-dimensional analysis. CONCLUSIONS: The strong correlations between the structural and mechanical properties obtained with the three techniques allowed us to validate the microCT technique used to characterize cortical bone microstructure. Porosity measurements might be of importance for clinicians and researchers to obtain a better understanding and evaluation of bone fracture in elderly patients.

Comparison of synchrotron radiation and conventional x-ray microcomputed tomography for assessing trabecular bone microarchitecture of human femoral heads.

Microcomputed tomography (microCT) produces three-dimensional (3D) images of trabecular bone. We compared conventional microCT (CmicroCT) with a polychromatic x-ray cone beam to synchrotron radiation (SR) microCT with a monochromatic parallel beam for assessing trabecular bone microarchitecture of 14 subchondral femoral head specimens from patients with osteoarthritis (n=10) or osteoporosis (n=4). SRmicroCT images with a voxel size of 10.13 microm were reconstructed from 900 2D radiographic projections (angular step, 0.2 degrees). CmicroCT images with a voxel size of 10.77 microm were reconstructed from 205, 413, and 825 projections obtained using angular steps of 0.9 degrees, 0.45 degrees, and 0.23 degrees, respectively. A single threshold was used to binarize the images. We computed bone volume/ tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th and Tb.Th*), trabecular spacing (Tb.Sp), degree of anisotropy (DA), and Euler density. With the 0.9 degrees angular step, all CmicroCT values were significantly different from SRmicroCT values. With the 0.23 degrees and 0.45 degrees rotation steps, BV/TV, Tb.Th, and BS/BV by CmicroCT differed significantly from the values by SRmicroCT. The error due to slice matching (visual site matching +/- 10 slices) was within 1% for most parameters. Compared to SRmicroCT, BV/TV, Tb.Sp, and Tb.Th by CmicroCT were underestimated, whereas Tb.N and Tb. Th* were overestimated. A Bland and Altman plot showed no bias for Tb.N or DA. Bias was -0.8 +/- 1.0%, +5.0 +/- 1.1 microm, -5.9 +/- 6.3 microm, and -5.7 +/- 29.1 microm for BV/TV, Tb.Th*, Tb.Th, and Tb.Sp, respectively, and the differences did not vary over the range of values. Although systematic differences were noted between SRmicroCT and CmicroCT values, correlations between the techniques were high and the differences would probably not change the discrimination between study groups. CmicroCT provides a reliable 3D assessment of human defatted bone when working at the 0.23 degrees or 0.45 degrees rotation step; the 0.9 degrees rotation step may be insufficiently accurate for morphological bone analysis.

The mode of bone conservation does not affect the architecture and the tensile properties of rat femurs.

The bone samples used in clinical and experimental trials must be the less damaged as possible to avoid alterations of their properties. However, the mode of storage might possibly alter the bone properties, particularly microarchitecture and strength. The aim of our study was to analyze the effects of deep-freezing and alcohol conservation techniques on the densitometric, microarchitectural and biomechanical parameters of rat femurs. The left femurs were elongated in uniaxial tension up to breakdown in order to calculate biomechanical parameters. The densitometric and microarchitectural properties of right femurs were evaluated using dual-energy X-ray absorptiometry and microcomputed tomography, respectively. Results showed no significant difference in the parameters investigated between deep-freezing, alcohol storage and fresh femurs when comparing each parameter separately. Therefore, one month storage in alcohol or deep-freezing seemed to induce no harmful effect on densitometric, microarchitectural and biomechanical parameters of rat femurs.

Alteration of trabecular bone under chronic beta2 agonists treatment.

PURPOSE: beta2 adrenergic agonists are widely used as doping agents. Their side effects on bone, especially microarchitecture, remain unknown. The purpose of this study was to evaluate the effects of chronic clenbuterol and salbutamol treatment on bones of growing rats. METHODS: Twelve-week-old Wistar female rats were divided into three groups: salbutamol (4 mg.kg(-1).d(-1)), clenbuterol (2 mg.kg(-1).d(-1)), and normal saline (0.5 mL.kg(-1).d(-1)) and treated for 6 wk. Proximal tibia and lumbar spine L4 were analyzed by absorptiometry and by 3D microcomputed tomography. Bending and compression tests were used to measure their mechanical properties. RESULTS: After 6 wk, the salbutamol and clenbuterol groups had lower bone mineral density (BMD) in the tibia, proximal tibia, and vertebrae. Trabecular number and bone volume for the vertebrae were lower in animals treated with clenbuterol (Tb.N: -14.31%, P < 0.001; BV/TV: -21.07%, P < 0.001) or salbutamol (TbN: -12.7%, P < 0.001; BV/TV: -19.7%, P < 0.001) than in controls. Mechanical properties of the tibia were affected by clenbuterol with a lower ultimate force (P = 0.02) and a trend in lower energy to ultimate force (P = 0.053). In vertebrae, salbutamol and clenbuterol induced lower ultimate force. Clenbuterol significantly increased muscle mass (+58.83%, P < 0.01) and reduced fat mass (-28.75%, P < 0.01) compared with controls +17.07 and -7.34%, respectively. CONCLUSION: This study shows a negative effect of clenbuterol and salbutamol on the mechanical properties and microarchitecture of trabecular bone. In the clenbuterol group it was notable that the bone loss contrasts with the anabolic effect on muscle mass. Clearly an increase of muscle mass with enhanced bone fragility augments the risk of fractures for humans or animals treated with beta2 agonists as part of a doping regimen.

A new anisotropy index on trabecular bone radiographic images using the fast Fourier transform.

BACKGROUND: The degree of anisotropy (DA) on radiographs is related to bone structure, we present a new index to assess DA. METHODS: In a region of interest from calcaneus radiographs, we applied a Fast Fourier Transform (FFT). All the FFT spectra involve the horizontal and vertical components corresponding respectively to longitudinal and transversal trabeculae. By visual inspection, we measured the spreading angles: Dispersion Longitudinal Index (DLI) and Dispersion Transverse Index (DTI) and calculated DA = 180/(DLI+DTI). To test the reliability of DA assessment, we synthesized images simulating radiological projections of periodic structures with elements more or less disoriented. RESULTS: Firstly, we tested synthetic images which comprised a large variety of structures from highly anisotropic structure to the almost isotropic, DA was ranging from 1.3 to 3.8 respectively. The analysis of the FFT spectra was performed by two observers, the Coefficients of Variation were 1.5% and 3.1 % for intra-and inter-observer reproducibility, respectively. In 22 post-menopausal women with osteoporotic fracture cases and 44 age-matched controls, DA values were respectively 1.87 +/- 0.15 versus 1.72 +/- 0.18 (p = 0.001). From the ROC analysis, the Area Under Curve (AUC) were respectively 0.65, 0.62, 0.64, 0.77 for lumbar spine, femoral neck, total femoral BMD and DA. CONCLUSION: The highest DA values in fracture cases suggest that the structure is more anisotropic in osteoporosis due to preferential deletion of trabeculae in some directions.

Anisotropy changes in post-menopausal osteoporosis: characterization by a new index applied to trabecular bone radiographic images.

Bone intrinsic strength is conditioned by several factors, including material property and trabecular micro-architecture. Bone mineral density (BMD) is a good surrogate for material property. Architectural anisotropy is of special interest in mechanics-architecture relations and characterizes the degree of directional organization of a material. We have developed anisotropy indices from the Fast Fourier Transform (FFT) on bone radiographs. We have validated these indices in a cross-sectional uni-center case-control study including 39 postmenopausal women with vertebral fracture and 70 age-matched control cases. BMD was measured at the lumbar spine and femoral neck. A fractal analysis of texture was performed on calcaneus radiographs at three regions of interest (ROIs), and the result was expressed as the H parameter (fractal dimension =H-2). The anisotropy evaluation was based on the FFT spectrum of these three ROIs extracted on calcaneus radiographs. On the FFT spectrum, we have measured the spreading angle of the longitudinal trabeculae called the dispersion longitudinal index (DLI) and the spreading angle of the transversal trabeculae called the dispersion transversal index (DTI). From the measured parameters, an anisotropy index was derived, and the degree of anisotropy (DA) calculated with DLI and DTI. We have compared the results from the vertebral fracture cases and control cases. The best distinction was obtained for the largest ROI located in the great tuberosity of the calcaneus for all parameters ( P <10(-4)).( )The DA parameter showed a higher value in vertebral fracture cases (1.746+/-0.169) than in control cases (1.548+/-0.136); P <10(-4), and the difference persisted after removal of the subjects with hormonal replacement therapy. The analysis of the receiver operating characteristics (ROC) has shown the best results with DA and Hmean: areas under curves (AUCs) respectively of 0.765 and 0.683, while AUCs associated to LS-BMD and FN-BMD were 0.614 and 0.591 lower, respectively. We determined the odds ratios (OR) by uni- and multivariate analysis. Crude ORs were respectively 3.91 (95% CI: 2.22-6.87) and 3.08 (95% CI: 1.72-5.52) for DA and Hmean. Crude ORs were respectively 1.71 (95% CI: 1.15-2.56) and 1.56 (95% CI: 1.05-2.31) for LS-BMD and FN-BMD. All ORs were statistically significant, and those associated to Hmean and anisotropy indices were higher than those of BMD measurements. From a multivariate analysis including anisotropy indices, Hmean, age and FN-BMD, the remaining significant ORs were respectively 6.33 (95% CI: 2.80-14.30) and 3.08 (95% CI: 1.48-6.37) for DA and Hmean. These data have shown that anisotropy indices on calcaneus radiographs can distinguish vertebral fracture cases from control cases. This analysis provides complementary information concerning the BMD and fractal parameter. These data suggest that we can improve the fracture risk evaluation by adding information related to the directional organization of trabecular bone derived from the FFT spectrum on conventional radiographic images.