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DOI: 10.1148/radiol.2293020558
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(Radiology 2003;229:921-928.)
© RSNA, 2003

Technical Developments

Excised Bone Structures in Mice: Imaging at Three-dimensional Synchrotron Radiation Micro CT1

Estela Martín-Badosa, PhD2, Daniel Amblard, PhD, Stefania Nuzzo, PhD, Abdelmajid Elmoutaouakkil, PhD, Laurence Vico, PhD and Françoise Peyrin, PhD

1 From the European Synchrotron Radiation Facility, BP 220, 38043 Grenoble, France (E.M.B., S.N., A.E., F.P.); LBTO, Equipe Mixte, Institut National de la Santé et de la Recherche Médicale, E9901 Faculté de Médecine, Saint-Etienne, France (D.A., L.V.); and CREATIS, UMR 5515 Centre National de la Recherche Scientifique, Villeurbanne, France (S.N., A.E., F.P.). Received May 10, 2002; revision requested July 25; final revision received February 26, 2003; accepted April 30. Supported by a Marie Curie Fellowship of the European Community program Human Potential under contract number HPMF-CT-1999–00194 and by grants from the European Research in Space and Terrestrial Osteoporosis group and Région Rhône-Alpes. Address correspondence to F.P. (peyrin@esrf.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and Methods
Results
Discussion
 REFERENCES
 
Bone microarchitecture and mineralization were determined at three-dimensional synchrotron radiation micro computed tomography in two inbred mice strains. Distal metaphysis of the left femur was imaged in three dimensions at 6.65 µm, whereas the right femur was analyzed with histomorphometry. Three-dimensional quantitative parameters of trabecular and cortical bone architecture were computed. C3H/HeJ@Ico mice had greater bone density and thicker trabeculae; greater cortical bone density, cortical thickness, and porosity; and greater mineralization than did C57BL/6J@Ico mice. The technique is well suited for assessment of trabecular and cortical bone in small animals and at the same time provides mineralization status in three dimensions.

© RSNA, 2003

Index terms: Animals • Bones, mineralization • Computed tomography (CT), three-dimensional, 40.12117 • Osteoporosis, 40.56 • Synchrotron


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and Methods
Results
Discussion
 REFERENCES
 
Mice models are increasingly recognized as powerful tools for medical research, especially for studies about genetics. The investigation of small animals by using medical imaging techniques may yield subsequent benefits in the exploitation of these models. However, most biomedical imaging devices have been optimized for human studies and have suboptimal spatial resolution for small animals (1). This currently motivates new developments in high-resolution imaging systems, such as micro computed tomography (CT), magnetic resonance (MR) imaging microscopy, and micro positron emission tomography.

In particular, because of the increasing prevalence of osteoporosis in developed countries (2), the interest in studies requiring the imaging of bone structures in small animals is expected to increase rapidly (3,4). Mouse models are particularly well suited for the study of bone loss and its genetic determinants (5). Current evaluation of bone loss most often is based on results of planar dual-energy x-ray absorptiometry. Although bone mass is an important determinant of bone strength, it does not take into account architectural changes in trabecular microstructure. Findings of a large number of experimental studies support the independent role of architecture in conferring strength to the trabecular network (6–9). In animal models, it is easy to perform an invasive bone biopsy and histomorphometry, which yield not only bone volume fraction but also microarchitectural parameters. Histomorphometry, however, has a fundamental limitation: Three-dimensional (3D) parameters are typically inferred from stereologic analysis of a limited number of two-dimensional (2D) anatomic sections. New imaging techniques based on CT and MR imaging enable 3D nondestructive analysis (10–12).

Results of the investigation of a rat model of osteoporosis with x-ray micro CT have been reported in the literature (13,14). In this last study, 3D micro CT, used with a voxel size of 24 µm, enabled the detection of bone loss earlier than did histomorphometry in a model of osteoporosis in rats sacrificed at different time points of the experimental design. The use of mice, with thinner bone structures, requires higher spatial resolution than is needed for imaging of rats. To this aim, the combination of synchrotron radiation (SR) with micro CT techniques possesses outstanding advantages (15,16). In this case, a high photon flux monochromatic x-ray beam extracted from a synchrotron beam replaces the standard x-ray beam with conventional micro CT devices. The main advantages of SR micro CT compared with standard micro CT are the following: The use of single x-ray beam energy prevents beam-hardening artifacts that can appear when a nonmonochromatic beam is used, the high photon flux enables the radiologist to obtain images with a high signal-to-noise ratio and a high spatial resolution in a reduced acquisition time, and the use of a nearly parallel beam enables exact CT reconstruction.

The device developed on beam-line ID19 at the European Synchrotron Radiation Facility, Grenoble, France, provides 3D images of bone samples with a spatial resolution as high as 1 µm with strong contrast and high signal-to-noise ratio (17). It has already been shown to be very accurate for quantifying human bone architecture (18,19).

The purpose of our study was to develop and evaluate a method based on SR micro CT for quantification of both the microarchitecture and the mineralization of trabecular and cortical bone in selected regions of interest (ROIs) in two strains of mice with different skeletal characteristics.


   Materials and Methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
Discussion
 REFERENCES
 
Animals
We used two strains of mice. The C3H/HeJ@Ico (hereafter, C3H) strain had a greater bone mineral density than did the C57BL/6J@Ico (hereafter, B6) strain, but both strains had a similar body weight and bone size (20). Ten male 16-week-old mice (Charles River; Iffa Credo, L’Abresle, France) of each strain were acclimatized for 1 week with standard conditions (temperature, 23°C ± 2; cycle, 12 hours of light and 12 hours of darkness). Animals were housed individually with free access to water and food pellets (rodent diet no. R03–25; UAR, Epinay/Orge, France). The procedure for the care and killing of the animals was in accordance with the European community standards regarding the care and use of laboratory animals (authorization no. 04827, Ministère de l’Agriculture, France).

Bone Histomorphometry
Right femora were excised, fixed, and dehydrated in absolute acetone by one of the authors (D.A.). They were then embedded in methylmethacrylate-based medium at low temperature according to a method developed in another investigation (21). Histologic sections of 7-µm thickness were removed from the femoral metaphyses along a longitudinal frontal plane for subsequent measurements according to conventional bone histomorphometry (22).

The ratio of bone volume to total tissue volume, expressed as a percentage and evaluated by using histomorphometry, was measured in the femoral distal cancellous metaphysis at the level of the secondary spongiosa within an ROI extent from the growth plate to a point 2.5 mm proximal to the growth plate. The metaphyseal junction of the growth plate was not yet closed, but the primary spongiosa was either too thin to be measured or absent. This measurement was performed in seven modified Goldner sections with an automatic image analyzer (Biocom; Paris, France). Structural indices were then calculated with histomorphometry in the same cancellous bone area: trabecular thickness (measured in micrometers), trabecular separation (measured in micrometers), trabecular number (per millimeter), and the ratio of bone surface to bone volume (per millimeter).

Coefficients of variation for these parameters, obtained in 10 measurements, were less than 2.2%.

Bone Sample Preparation for 3D Measurements
For each strain, the measurements were performed on the 10 left femora of the same mice that were used for histomorphometric evaluations. The same author as noted before carefully excised the femora and placed them in 70% ethanol in Eppendorf 0.5-mL microtubes. The distal end of each femur was blocked in the conical bottom of the microtube, and the proximal end of the femur was immobilized with a propylene ring tightly placed between the bone and the tube. This sample holder was hermetically closed at the top to prevent ethanol evaporation and vertically glued on the rotating platform of the experimental setup.

Image Acquisition: SR Micro CT
Images were obtained by using SR micro CT at the ID19 beam line of the European Synchrotron Radiation Facility. In the acquisition system, a large quasi-parallel and very intense monochromatic SR beam is used. The scheme of the experimental setup is shown in Figure 1. The sample is placed on a rotating platform and partially stops the incident monochromatic beam. The number of transmitted photons is measured with a 2D detector, which consists of an x-ray–visible 5-µm-thick light converter (gadolinium oxysulfide doped with terbium scintillator), light optics for magnification of the image, and a charge-coupled device camera (Frelon camera) developed at the European Synchrotron Radiation Facility in Grenoble, France (23). The camera is 14-bit dynamic and has a 1,024 x 1,024-pixel chip. The pixel size is 19 µm, but because of optical magnification, effective pixel sizes between 10 µm and less than 1 µm can be chosen, depending on the optics used.



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  		Figure 1. SR micro CT experimental setup. Different elements of 3D acquisition device from left to right: monochromatic x-ray beam extracted from SR, sample stage, and 2D detector.

 
In our experiment, the pixel size was fixed to 6.65 µm, which seemed appropriate to image trabeculae, which were estimated from histologic data to have a thickness of approximately 20–30 µm, in mice. Because of the diameter of the total sample, which was 3 mm or smaller compared with typical 10-mm human bones that are usually imaged with a beam energy between 24 and 30 keV, the beam energy was lowered. A beam energy of 18 keV was found to provide suitable contrast. For each sample, 900 radiographic images (or projections) with 1,024 x 1,024 pixels were acquired at different angles of view between 0° and 180°. According to the design of the experiment, the photon flux incident on the sample was in the order of 6 x 109 photons/sec/mm2, and the total acquisition time was approximately 20 minutes per sample. A flat field correction was performed on each rough radiographic image to correct the spatial fluctuations of the x-ray beam and the inhomogeneity of the individual pixel of the detector (17). Figure 2, A, shows a corrected mediolateral projection of one femur sample from a mouse of the C3H strain.



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  		Figure 2. Representative images of C3H mouse. A, Mediolateral radiograph shows distal part of the femur. B, Posteroanterior 3D reconstructed volume shows selected region of distal part of the femur to be analyzed.

 
Image Reconstruction
After radiographic data acquisition, CT reconstruction was performed by means of a 3D filtered back-projection algorithm to retrieve the 3D bone structure image. For each sample, the reconstruction was limited to a 512 x 512 x 600-voxel subvolume, which corresponded to a cylinder with a diameter of 6.8 mm and a height of 4 mm, and included the distal femur above the growth cartilage. The reconstruction time was approximately 25 minutes per sample. The reconstructed values that corresponded to the linear attenuation coefficient within the femur were between 0 and 15 cm-1 and were quantified by using 256 image gray levels.

Separation of Cortical and Trabecular ROIs
Because of good contrast, bone was easily segmented from marrow or background with simple thresholding. The same threshold was used for all samples. Then the ROI to be analyzed was defined for all samples. For this purpose, one of the authors (E.M.B.) selected the bottom of the ROI above the cartilage of the growth plate for each sample. Then, an ROI with a height of 2.35 mm (354 voxels) was automatically extracted for all samples. The location of the ROI is illustrated in Figure 2, B, as the subvolume between the two horizontal sections.

This ROI that contained both cortical and trabecular bone was further automatically processed to separate the two components. This task could not be simply performed with operations that were based on gray levels, since both bone structures were in the same range of attenuation. However, the cortical envelope, the external shell surrounding the trabecular bone, is much more compact than is the trabecular bone (24). Thus, we developed a customized algorithm for identification of the cortical envelope that was based on geometrical considerations. The process was mainly based on an iterative filling procedure. The exterior region was scanned until bone was reached and filled with a constant gray-level value. Then, the same procedure was used to label the cortical region with a different gray-level value, starting from the exterior cortical border and stopping when the marrow structure (black regions) was reached.

The method was successfully applied to all samples. After separation, the two components were independently processed. Figure 3, A and B, shows 3D-rendered images of trabecular bone for the C3H and B6 mouse strains, respectively, whereas Figure 3, C and D, shows cortical bone for each strain after separation.



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  		Figure 3. Images show 3D displays of ROIs extracted from reconstructed bone volume. Left column: Images in C3H mice. Right column: Images in B6 mice. A, B, Trabecular ROIs. C, D, Cortical ROIs. E, F, Pores in cortical ROIs.

 
Trabecular Microarchitecture Analysis
The quantification of trabecular structures includes the extraction of parameters from the 3D trabecular bone images that were previously obtained. Different measurement methods are available. The first method relies on histomorphometry and provides parameters derived from an assumption in regard to the geometry of bone (eg, parallel plate model) (25). Similar parameters may be calculated section by section throughout the volume or directly in three dimensions by using a 3D mean intercept length method (26). The second method includes computation directly on the 3D image model-independent parameters, and thus no prior assumption was made about bone geometry (27). We used the latter approach to compute the following 3D parameters on the trabecular ROI: bone volume to total tissue volume (expressed as a percentage), bone surface to bone volume (per millimeter), trabecular thickness (expressed in micrometers), trabecular number (per millimeter), and trabecular separation (expressed in micrometers). In addition, the 3D connectivity density (per cubic millimeter) was measured from the computation of the 3D Euler number (28). The degree of anisotropy was computed as the ratio between the maximum and the minimum radii of the mean intercept length ellipsoid (26).

Cortical Analysis
Similar techniques were applied to quantify the cortical ROIs. Parameters were estimated from 3D measurements. First, the ratio of cortical bone volume to total tissue volume was calculated and expressed as a percentage. Then the cortical thickness (expressed in micrometers) was estimated by using the same model-independent technique that was used for measuring trabecular thickness. Although it is not the conventional use of this method, this method may be used to quantify the thickness of any 3D shape since no assumption is made regarding the structure to be quantified. The cortical connectivity density (per cubic millimeter) also was measured by using the same method as was used for the trabecular connectivity density: The connectivity was calculated from the Euler number of the cortical bone volume (28) and was normalized to the cortical bone volume to obtain a density measurement. Total tissue volume includes some natural porosity, which is included in the determination of the ratio of cortical bone volume to total tissue volume. We also examined the structure and size of these pores in the cortices. Figure 3, E and F, illustrates 3D-rendered images of pores in the cortical shell for the C3H and B6 mouse strains, respectively. Cortical porosity was defined as the volume of pores divided by the volume of cortical bone and was expressed as a percentage. The distribution of pore thickness was calculated by using the same direct method as was used for trabecular thickness. The thickness of pores (expressed in micrometers) was then estimated as the median of this distribution.

Mineralization Analysis
Because of the technical characteristics of the SR micro CT setup (ie, monochromaticity, high photon flux, and parallel beam), the images also were used quantitatively to assess the mineral content in bones. Indeed, the gray levels in the 3D images, which are representative of the linear attenuation coefficient of the samples (expressed in centimeters), are not uniform within bone tissue. The reconstructed gray levels of the SR micro CT image were related to the degree of mineralization in bone by using a theoretical relationship validated with experimental data (29).

The method was applied to both the cortical and trabecular ROIs that were determined previously. The histogram of the degree of mineralization normalized by the bone volume of the sample was calculated by using the correspondence between the gray levels and the degree of mineralization. Then, for each ROI, the degree of mineralization was estimated as the median value of the histogram. The degree of mineralization in both the trabecular and cortical bone ROIs was estimated. The mean value of each strain was computed as the average of the median results for the individual mouse.

Data Analysis
The parameters were computed separately for each mouse of the C3H (n = 10) and B6 (n = 10) strains. Data are presented here as the mean ± SD within each strain.

Statistical analyses were performed by using software (StatView 5.0 for Windows; Abacus Concepts, Berkeley, Calif). Comparisons between C3H and B6 strains were determined by using a Mann-Whitney nonparametric test. We considered that differences between the strains were statistically significant with P < .05.

To compare trabecular metrics calculated in three dimensions from micro CT volumes or in two dimensions from histologic findings, we computed Spearman rank correlation coefficients, or {rho}; a difference with P < .05 was considered significant.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
Discussion
 REFERENCES
 
Trabecular Analysis
Table 1 shows the mean values and SDs of the trabecular microarchitectural parameters measured by using SR micro CT in mice of both strains. The distribution of some parameters that were estimated from 3D measurements (ie, ratio of bone volume to total tissue volume, ratio of bone surface to bone volume, trabecular number, trabecular thickness, trabecular separation, and trabecular connectivity density) within each strain are illustrated in the box plots of Figure 4.


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  		TABLE 1. Trabecular Bone Parameters Estimated from Direct 3D Measurements and Histomorphometric Parameters

 


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  		Figure 4. Box plots for trabecular bone mass and microarchitectural parameters (all estimated from 3D measurements). A, Ratio of bone volume to total tissue volume (BV/TV*). B, Ratio of bone surface to bone volume (BS/BV*). C, Trabecular thickness (Tb.Th*). D, Trabecular separation (Tb.Sp*). E, Trabecular number (Tb.N*). F, Trabecular connectivity density (ConnD*). G, Degree of anisotropy (DA). Horizontal lines in each box from top to bottom indicate 25th, 50th, and 75th percentiles. Error bars indicate 10th and 90th percentiles; dots correspond to minimum and maximum values. Dagger denotes that differences between C3H and B6 strains are significant (P < .05).

 
These results demonstrate significant differences between mice of the C3H and B6 strains for a number of parameters that were estimated from the 3D measurements, namely, ratio of bone volume to total tissue volume, ratio of bone surface to bone volume, trabecular thickness, and trabecular connectivity density and for degree of anisotropy. The ratio of bone volume to total tissue volume estimated from the 3D measurements was 38% higher in mice of the C3H strain than it was in those of the B6 strain, whereas the ratio of bone surface to bone volume estimated from the 3D measurements was 27% lower. Mice of the C3H strain had thicker trabeculae than did those of the B6 strain, with a 61% relative variation, and the trabecular connectivity density estimated from the 3D measurements was 29% lower. Mice of the C3H strain were more anisotropic than those of the B6 strain with a degree of anisotropy 9% higher. Values for both the number of trabeculae estimated from the 3D measurements and the separation between them were similar for both strains.

Comparison with Histomorphometry
Table 1 also includes the results of trabecular bone and structural parameters measured by using histomorphometry. The conclusions regarding the statistical difference between strains are the same when using histomorphometry or SR micro CT, although the absolute values of parameters are different. The ratio of bone volume to total volume evaluated with histomorphometry was 40% higher for mice of the C3H strain than it was for those of the B6 strain, and the ratio of bone surface to bone volume evaluated with histomorphometry was 27% lower. For trabecular thickness, variations from histologic measurements between strains are less important; for mice of the C3H strain, trabecular thickness evaluated with histomorphometry was 37% higher than it was for mice of the B6 strain. Trabecular separation and trabecular number evaluated with histomorphometry were similar for both strains.

Table 2 shows the Spearman rank correlation coefficients between trabecular metrics computed in three dimensions from micro CT volumes or in two dimensions from histologic findings. The results of these two methods correlate well for the determination of parameters as follows: the ratio of bone volume to total tissue volume ({rho} = 0.756; P = .001), the ratio of bone surface to bone volume ({rho} = 0.770; P < .001), and trabecular thickness ({rho} = 0.808; P < .001). However, the measurements of trabecular separation and trabecular number with both methods did not show a significant correlation.


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  		TABLE 2. Correlation between Histomorphometric and 3D Parameters

 
Cortical Analysis
The values of cortical parameters for each strain and the statistical differences between strains are indicated in Table 3. Figure 5 includes 2D plots that show a combination of a pair of cortical parameters for each mouse and illustrates how the C3H and B6 strains can be discriminated. Relative cortical volume and cortical thickness were significantly greater for mice of the C3H strain than they were for those of the B6 strain (84% for the ratio of cortical bone volume to total tissue volume and 20% for cortical thickness, both of which were estimated from 3D measurements). Filling the pores in the cortical volumes before measuring the ratio of cortical bone volume to total tissue volume estimated from 3D measurements did not modify the statistical difference between the groups. The connectivity density was similar for both strains. Cortical porosity was 87% greater in mice of the C3H strain. The thickness of cortical pores also was significantly higher in mice of the C3H strain than it was in those of the B6 strain (difference between the two strains, 41%).


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  		TABLE 3. Cortical Bone Parameters Computed from Direct 3D Methods

 


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  		Figure 5. Two-dimensional plots of cortical parameters in C3H ({bullet}) and B6 ({blacktriangleup}) mice. Two strains of mice can be differentiated when they are described with a pair of cortical parameters (all estimated from 3D measurements). A, Cortical thickness (Ct.Th*) and cortical porosity (Ct.Por*). B, Relative cortical volume, expressed as the ratio of cortical bone volume to total tissue volume (CtV/TV*), and cortical connectivity density (Ct.ConnD*).

 
Mineralization Analysis
Figure 6 illustrates the representative distributions of the degree of mineralization in the trabecular and cortical ROIs for both strains. They were obtained by means of summation of the number of occurrences of each degree of mineralization and of normalization with the total number of events. The linear absorption coefficient was in the range of 7.5–13.0 cm-1, which corresponded to a range of 0.8–1.5 g of hydroxyapatite per cubic centimeter of bone. Table 4 reports the mean and SD of the degree of mineralization for both strains in the trabecular and cortical ROIs. Cortical bone appears more mineralized than does trabecular bone for both strains, since the corresponding histograms are shifted toward the right. In addition, the bone of mice of the C3H strain was significantly more mineralized than was the bone of mice of the B6 strain, both in trabecular (14%) and cortical (7%) bone.



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  		Figure 6. Graph shows differences between C3H and B6 strains of mice in terms of mineralization. Mean histogram of each strain is plotted: Solid curves correspond to C3H strain; and dotted curves, to B6 strain. Dark curves correspond to trabecular (trab) ROIs; and light curves, to cortical (cort) ROIs.

 

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  		TABLE 4. Degree of Mineralization in Trabecular and Cortical Bone

 

   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
Discussion
REFERENCES
 
Three-dimensional SR micro CT was used to investigate bone architecture in two different strains of mice. After image acquisition, 3D image processing techniques were developed to fully exploit 3D data. An automatic method was designed to extract and separate trabecular and cortical ROIs, which allowed the analysis of both regions independently. Contrary to conventional approaches, all quantitative parameters were computed directly from 3D data without any model assumption. Moreover, the very high spatial resolution of SR micro CT enabled a quantification of the porous network in cortical bone, which, to our knowledge, was accomplished here for the first time in three dimensions with micro CT data. Finally, the physical characteristics of the SR micro CT device made it possible to quantify the degree of mineralization of bone. To the best of our knowledge, such information has not been previously obtained with standard micro CT, since the polychromaticity of x-ray beams used at standard micro CT makes this task challenging.

Our results indicate that the two strains have very different microstructural characteristics in both trabecular and cortical regions. Findings in previous works (20,30) have shown that these two inbred strains of mice have differences in peak bone densities and bone structural parameters, but to our knowledge, investigations have never been performed in the femoral region at this spatial resolution. We chose to investigate the distal femoral metaphysis, which offers a large ROI and is a site rapidly challenged in bone loss models (5) of 4-month-old mice that at this stage have a mature skeleton (20).

With regard to trabecular microarchitecture, we found that mice of the C3H strain had greater bone density and thicker trabeculae than did those of the B6 strain, whereas the ratio of bone surface to bone volume was significantly lower. No differences were observed for the number of trabeculae and the separation between them. Our conclusions differ from those previously reported (30) about trabecular microarchitecture on lumbar vertebrae by using micro CT with a voxel size of 17 µm. In the previous study, a lack of trabecular structure in the C3H strain was observed, with a similar trabecular thickness in both strains. This may suggest a heterogeneity between bone sites, which also has been demonstrated in different strains of rats (31).

The parameters computed with 2D histologic sections yielded the same conclusions regarding the differences between strains. Trabecular bone mass parameters (ratio of bone volume to total tissue volume and ratio of bone surface to bone volume) measured by using SR micro CT correlated well to those measured by using standard histomorphometry. A previous study, in humans, has shown the same results when histologic findings were compared with those at conventional x-ray micro CT (32). Nevertheless, architectural parameters such as trabecular number and trabecular separation were not correlated. These differences may arise from a number of factors. First, histomorphometry and SR micro CT were performed with different samples; in each mouse, the right femur was used for histomorphometry, whereas the left one was imaged by means of SR micro CT. Second, the measurements were determined with 2D and 3D data, respectively, and the parameters were computed with different methods. With the approach used for computation of the 3D model-independent parameters, no prior assumption was made about the bone geometry. Thus, we believe that the resulting 3D parameters are more faithful, since they avoid the parallel plate model assumed in histomorphometry and are computed with a larger data set (3D volume instead of a limited number of sections). In addition, compared with histomorphometry, the 3D nature of SR micro CT data permits the estimation of connectivity and anisotropy, which are necessarily biased when estimated with 2D images. Both parameters appeared to be significantly different when the two strains were compared, with the B6 strain having a more connected and isotropic structure.

Our data allowed measurement of structural and topological parameters in cortical bone. We found that mice of the C3H strain had significantly greater cortical bone density, which may be related to the greater cortical area and volume previously described (30,33). The greater cortical thickness found in mice of the C3H strain is also in agreement with previous findings (30,33). However, the absolute values obtained in our study were lower, which may be related to the location of the analyzed ROIs. We acknowledge that results of the present study did not allow correlation of the results of the analysis of cortical bone with those of SR micro CT and histomorphometry, but this approach would have required a different protocol with transverse sections.

Cortical porosity was significantly greater in mice of the C3H strain than it was in those of the B6 strain, which confirmed the visual observation. We noticed very wide distributions of pore thickness, meaning that pores of quite different sizes were present in the cortical bone. Considering the median value of these distributions as indicative of the size of pores, significantly smaller sizes were obtained for the B6 strain. The quantification of cortical porosity with micro CT data has not been reported, to our knowledge—certainly because of the lack of spatial resolution. Even in the present study, one can observe that the smaller pores in mice of the B6 strain are close to the resolution limit, and the results might be distorted. More accurate results could be obtained with the higher spatial resolution available with our system but at the expense of reducing the field of view and, thus, the analyzed ROI.

Thanks to SR monochromaticity, a quantitative analysis of the degree of mineralization also was performed. The bone tissue in mice of the C3H strain was significantly more mineralized, which is in agreement with the conclusions reported with techniques that were based on back-scattering electron imaging (30) and on peripheral quantitative CT (20). However, our technique allowed separate measurements of the degree of mineralization in cortical and trabecular bone regions. The lower degree of mineralization observed in trabecular tissue in mice of both strains may be related to the fact that the remodeling is higher in trabecular bone than it is in cortical bone. In mice of the C3H strain, the higher mineralization in the femoral shaft could be related to the superior biomechanical properties reported for that site (30). However, the relationship between the degree of mineralization of bone and its biomechanical properties is still an open research topic, and other characteristic features of cortical bone also could be taken into account. For that purpose, our method possesses the advantage of providing simultaneous measurements of different properties in the same bone sample.

SR micro CT was used for a detailed examination of mice bone samples both in terms of microarchitecture and mineral content. Image analysis techniques that provided 3D model-independent parameters in both trabecular and cortical ROIs allowed highlighting of the differences between two strains of mice. The technique presented here, with higher spatial resolution and signal-to-noise ratio than those of standard micro CT, appears to be particularly well suited for high-resolution characterization (ie, bone mass, microarchitecture, and degree of mineralization) of the bones of small animals.


   ACKNOWLEDGMENTS
 
E.M.B. thanks the Ministerio de Educación y Cultura, Spain, and the European Commission for their postdoctoral grants. The images were acquired at the European Synchrotron Radiation Facility at experiment LS1393 (November 13–15, 1999). We acknowledge the ID19 group for help during data acquisition. We thank Susan Bloomfield, PhD, for assistance with the English language and IFFA-CREDO (l’Arbresle, France) for their sponsorship.


   FOOTNOTES
 
2 Current address: Lab d’ Òptica, Department of Applied Physics and Optics, University of Barcelona, Spain. Back

Abbreviations: ROI = region of interest, SR = synchrotron radiation, 3D = three-dimensional, 2D = two-dimensional

Author contributions: Guarantors of integrity of entire study, F.P., L.V.; study concepts and design, F.P., L.V.; literature research, D.A., E.M.B.; experimental studies, D.A., L.V.; data acquisition, all authors; data analysis/interpretation, E.M.B., A.E., F.P.; statistical analysis, E.M.B., L.V.; manuscript preparation and editing, E.M.B., F.P.; manuscript definition of intellectual content, E.M.B., F.P., L.V.; manuscript revision/review and final version approval, all authors


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
Discussion
 REFERENCES
 

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