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J Appl Physiol 100: 274-280, 2006. First published September 1, 2005; doi:10.1152/japplphysiol.00495.2005
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Monochromatic synchrotron radiation µCT reveals disuse-mediated canal network rarefaction in cortical bone of growing rat tibiae

Takeshi Matsumoto,1 Masayuki Yoshino,1 Takahisa Asano,2 Kentaro Uesugi,3 Masahiro Todoh,4 and Masao Tanaka1

1Division of Bioengineering, Osaka University Graduate School of Engineering Science, Toyonaka; 2Department of Nuclear Medicine, Kawasaki Medical School, Kurashiki; 3SPring-8/Japan Synchrotron Radiation Research Institute, Kouto; and 4Division of Mechanical Science, Hokkaido University Graduate School of Engineering, Sapporo, Japan

Submitted 29 April 2005 ; accepted in final form 29 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The purpose of this study was to demonstrate the ability of computed microtomography based on monochromatic synchrotron radiation (SRµCT) in microstructural analysis of cortical bone. Tibial diaphyses of growing rats (14 wk, n = 8) undergoing unilateral sciatic neurectomy 8 wk before study were imaged with spatial volume resolution of 5.83 x 5.83 x 5.83 µm3 by SRµCT (20 keV) at the synchrotron radiation facility (SPring-8). Reconstructed image data were translated into local mineral densities by using a calibrated linear relationship between linear absorption coefficients and concentrations of homogeneous K2HPO4 solution. Pure bone three-dimensional images, produced by simple thresholding at a bone mineral density of 0.82 g/cm3, were analyzed for macro- and microscopic structural properties. In neurectomized hindlimbs, cortical canal network rarefaction as well as bone atrophy were found. The former was characterized by 30% smaller porosity, 11% smaller canal density in transverse section, and 38% smaller canal connectivity density than those in contralateral bone. On the other hand, no difference was found in bone mineral density between neurectomized and intact hindlimbs (1.37 vs. 1.36 g/cm3). In conclusion, SRµCT is a promising method for the three-dimensional analysis of cortical microstructure and the degree of mineralization in small animals.

computed microtomography; cortical canal network; bone mineralization; unilateral sciatic neurectomy

BONE MICROSTRUCTURE HAS BEEN studied with a focus on cancellous bone, because its high turnover rate or high vulnerability to bone loss is of great clinical interest. A further impetus to the study on cancellous bone has been the availability of computed microtomography based on a microfocus X-ray source (µCT), allowing three-dimensional (3D) analysis of trabecular microstructure (3, 11, 29). On the other hand, only a few studies have reported on cortical microstructure or vascular canal network structure, despite the fact that it plays important roles in the bone remodeling process (25, 40) and cortical microcirculation (5, 20).

The insufficient spatial resolution of laboratory µCT may be a major barrier to 3D analysis of cortical microstructure, especially in small animals, which serve as a variety of bone disease models. Although two-dimensional (2D) histomorphometry or 3D reconstruction based on serial histological sectional images could provide information on cortical microstructure (37, 42), such techniques are subject to deformation artifacts and are time-consuming. Besides, anisotropic properties of vascular canal network make it difficult to apply 2D image-based analyses.

Synchrotron radiation (SR) combined with µCT (SRµCT) has opened up new possibilities in 3D analysis of cortical microstructure (1, 10, 13, 36). The SR properties of natural collimation and extremely high light intensity, even after monochromatization, enable SRµCT to reconstruct the highly resolved 3D image with a high signal-to-noise ratio. Furthermore, the availability of monochromatic X-rays eliminates beam-hardening artifacts and, therefore, allows image quantification for determining local bone mineralization (32, 33, 41). Laboratory µCT, although its resolving power has increased up to the acceptable level for the 3D reconstruction of cortical microstructure (8, 46), lacks such advantages specific to monochromatic SRµCT.

To date, the ability of SRµCT has been demonstrated mainly in cancellous bone analysis (2, 16, 19, 32, 35, 38, 41); however, there have been very few SRµCT studies of cortical bone. A detailed description of cortical canal pore structure would be valuable for understanding cortical microcirculation, because its vascularity is closely related to canal network structure (22). Furthermore, cortical bone mineralization, along with measures of canal geometry, is crucial for prediction of bone strength (39, 48). Thus the present study was aimed to demonstrate the utility of SRµCT in analysis of cortical microstructure and mineralization by measuring growing rat tibiae subjected to sciatic neurectomy, which induce bone, muscle, and vascular atrophies in disused hindlimbs (12, 15, 28, 51).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experiments were conducted according to the guiding principles of the American Physiological Society and with an approval of the Animal Research Committee of Osaka University Graduate School of Engineering Science.

Sample preparation.   Six-week male Wistar rats (140–150 g, n = 8) underwent unilateral sciatic neurectomy of the left hindlimb under anesthetization with intraperitoneal administration of pentobarbital sodium (50 mg/kg). The contralateral hindlimb was left intact as the internal control. After the operation, the rats were housed singly with normal cage activity and allowed free access to standard lab chow and tap water. Eight weeks later, all rats, weighing 350–390 g, were killed with a saturated KCl solution administered through a femoral vein under pentobarbital sodium anesthesia (50 mg/kg ip). Both tibiae in operated (OP) and intact (non-OP) contralateral hindlimbs were excised, cleaned of soft tissue, and soaked in 70% ethanol for 7 days. Then the 4-mm-long diaphyses immediately proximal to the tibio-fibula junctions were cut out by using a diamond-saw cutter (South Bay Technology, SBT650).

SRµCT.   Sample imaging by SRµCT was performed at beamline BL20B2 in the SR facility, Super Photon Ring-8GeV (SPring-8) in Harima, Japan. The detailed experimental arrangement for utilizing monochromatic SR beam has been described in earlier works (16, 41). In the present study, we set the X-ray energy to 20 keV with the relative energy spread of 0.01%. The photon flux incident on the sample was >1 x 109 photons·s–1·mm–2, and the X-ray size for sample irradiation was 5 mm in height and 75 mm in width.

The samples were encapsulated in a polyethylene tube and mounted on a goniometer, allowing high-precision translations and rotations. A high-resolution X-ray detector, consisting of a fluorescent screen, a beam monitor (Hamamatsu Photonics, AA40P), and a cooled charge-coupled device camera (Hamamatsu Photonics, C4880–10-14A) was used for radiographic imaging. The fluorescence screen, in which 1-µm particles (P43, Gd2O2S:Tb+) were filled at a 50% volume fraction over the thickness of 10 µm, is applicable to SRµCT with a few micrometer resolution. The photon flux transmitted through the object was transformed into a visible image with a magnification factor of 2. The viewing field of 4.0 mm in height and 5.8 mm in width was resolved into 5.83-µm pixels with 14-bit resolution. For each sample, 360 radiographic images were acquired over an angular range of 0–180° in 0.5° steps. Data-acquisition time was ~1.5 h/sample. Reconstruction was made with a 2D filtered backward projection algorithm on a Windows PC, providing 700 contiguous 2D images composed of 1,000 x 1,000 cubic voxels of 5.83-µm size in each. Then the reconstructed images were redigitized with 8-bit resolution, proportional to the linear absorption coefficient. Commercial volume-rendering software (Studio Pon, FORGE) running on Windows PC was used for the 3D visualization of canal network.

Calibration.   The use of monochromatic SR allows the quantification of bone hydroxyapatite (HAp) because it is free from beam-hardening artifacts. Assuming that the X-ray absorption of bone is described as a two-phase mixture (HAp and light elements), the following linear relationship holds between the linear absorption coefficient (µbone) and the HAp density ({rho}HAp) of bone:

(1)
To determine A and B, dipotassium hydrogen phosphate (K2HPO4) water solutions of various concentrations were measured by SRµCT under the same experimental condition for bone samples (32, 33). Only 6% difference of mass absorption coefficient between K2HPO4 and HAp for the 20-keV X-ray [National Institute of Standards and Technology (NIST) database] validates the use of K2HPO4 solution as a substitute for the HAp powder solution. Furthermore, highly homogeneous K2HPO4 solution is more suitable for calibration than HAp powder solution. Thus the linear regression between the linear absorption coefficient and the concentration of K2HPO4 solution provides the estimates of A and B. To examine the validity of Eq. 1 with the experimentally determined values of A and B, a synthetic auditory ossicle (Pentax, Apaceram AB07-T) composed of uniformly dense HAp (3.12 g/cm3) was measured by SRµCT, and the experimental and predicted linear absorption coefficients were compared.

Image analysis.   The central portion of each sample with a height of 2,332 µm (i.e., 400 contiguous transverse sections) was analyzed for canal network structure. Before determination of bone structural indexes, SRµCT images were binarized with simple thresholding to differentiate pure bone from background, medulla, and canal pores. The threshold value was determined through the comparisons between 2D SRµCT images binarized at various threshold values and the image by light micrography (Nikon, Eclipse E400 and CoolPix 990) of the non-decalcified sliced sample showing the same transverse section.

As macroscopic structural indexes, cortical tissue volume (CTV), cortical transverse sectional area (CTA), and medullary transverse sectional area (MA) were calculated, where the latter two were presented as the averages over 400 transverse sections. Trabeculae protruding into medulla, which occupied negligible medullary space in the present bone tissue specimens, were included in CTV and CTA.

Indexes characterizing cortical microstructure were as follows: cortical porosity or canal volume fraction per CTV, mean cross-sectional area and number density of canals penetrating transverse sections per CTA (averaged over 400 transverse sections), number density of canal bifurcations or junctions of three canal segments (CaBf/CTV), number density of canal links (CaLn/CTV), and number density of canal loops (CaLp/CTV). In the process of transverse sectional image scanning, included in the count of CaBf were canal bifurcations where both branching canals extended beyond three contiguous slices. In the count of CaLn, branching canals that met other canals ahead of their counterpart branch were included. The first Betti number of 1-complex topologically equivalent to canal network was defined as CaLp, which was given by the Euler-Poincaré relation. To take into account the highest degree of canal connectivity, the 26-adjacency was adopted for counting CaBf, CaLn, and CaLp.

In the evaluation of bone mineralization based on Eq. 1, the distribution of {rho}HAp was determined from the distribution of µbone in pure bone. Comparisons of the mean value and the coefficient of variation (SD/mean) of {rho}HAp were made between OP and non-OP tibiae.

Statistics.   Differences between OP and non-OP tibiae were assessed with Wilcoxon matched-pairs signed-rank test. A value of P < 0.05 was considered statistically significant. Data are represented as means (SD).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Figures 1, A and B, show, respectively, the 2D reconstructed image of a transverse section of capillary glass tubes containing K2HPO4 solution at different concentrations and the profile of gray level along the l-l' line drawn in A. The almost flat profile of gray level implies no beam-hardening artifact in the present SRµCT. In Fig. 1C, linear absorption coefficients, which averaged over the entire 2D region within each capillary tube, are plotted as a function of K2HPO4 concentration. The linear regression provided the following approximation for the relationship between {rho}HAp and µbone:

(2)
with r2 > 0.999. The linear absorption coefficient of the HAp ossicle predicted by substituting its uniform density ({rho}HAp = 3.12 g/cm3) into Eq. 2 differed by only 2.3% from the value of 17.4 cm–1 measured by SRµCT.



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  		Fig. 1. Reconstructed image of a transverse section of capillary glass tubes containing K2HPO4 water solutions of various concentrations (A) and the profile of gray level along the l-l' line (B). Concentrations of K2HPO4 are 0.10, 0.28, 0.44,0.61, and 0.76 g/cm3. The flat profile of gray level shows no beam-hardening artifact. C: a highly linear relationship was found between concentrations (c) and linear absorption coefficients of K2HPO4 solutions (µ) determined by computed microtomography using monochromatic synchrotron radiation (SRµCT).

 
Figure 2 shows the 2D reconstructed image of a non-OP tibial transverse section (A) and the relative distribution of linear absorption coefficient in the entire area of A, including background, medulla, and canal pores (B). Simple thresholding is found to operate well because the distribution of linear absorption coefficient shows the clear peak corresponding to HAp. In Fig. 3, a portion of cortical transverse section imaged by SRµCT (A), its binary image using a threshold value of 5.3 cm–1 (B), and a micrograph of the same transverse region (C) are shown. Vascular canals observed in the micrograph are well reproduced in the binary image. This threshold value, corresponding to the {rho}HAp of 0.82 g/cm3, according to Eq. 2, was used for every image binarization.



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  		Fig. 2. Reconstructed image of an intact (non-OP) tibial transverse section in an intact right hindlimb (A) and its relative distribution of linear absorption coefficient (B). The threshold value for pure bone segmentation was set to 5.3 cm–1 for all SRµCT images. Bar = 200 µm.

 


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  		Fig. 3. Portion of tibial cortical transverse section imaged by SRµCT (A), SRµCT and subsequent binarization using a threshold value of 5.3 cm–1 (B), and light micrography (C). Bar = 50 µm.

 
Figure 4 provides volume-rendered 3D displays of a pair of non-OP (A) and OP (B) tibiae, generated from binarized 2D reconstructed images. Neurectomy induced bone atrophy and canal network rarefaction simultaneously. Both CTV and CTA were smaller, whereas MA was slightly larger, in OP than in non-OP tibiae [8.34 (SD 0.32) vs. 10.71 mm3 (SD 0.49), 3.58 (SD 0.14) vs. 4.59 mm2 (SD 0.21), and 1.22 (SD 0.10) vs. 1.14 mm2 (SD 0.08), P < 0.05]. The index values for cortical microstructure of OP and non-OP tibiae are compared in Table 1. All indexes showed significant differences, indicating reduced canal network in OP tibiae.



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  		Fig. 4. Volume-rendered images of a pair of left non-OP (A) and right operated (OP; B) tibiae, based on 200 contiguous two-dimensional binary reconstructed images (1.166-mm thickness in total). The enlarged anterior portions are depicted below: interconnecting canals are red, and bifurcation points are blue.

 

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  		Table 1. Cortical microstructural properties

 
In Fig. 5, the relative distributions of {rho}HAp (>0.82 g/cm3) are depicted for a pair of OP and non-OP tibiae. Two distributions are almost the same, indicating no disuse effect on mineral density distribution. Actually, no difference was found in both {rho}HAp [1.37 (SD 0.01) vs. 1.36 g/cm3 (SD 0.01)] and the coefficients of variation of {rho}HAp [0.085 (SD 0.002) vs. 0.088 (SD 0.002)] between OP and non-OP tibiae.



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  		Fig. 5. Relative distributions of hydroxyapatite (HAp) density for a pair of OP (thick shaded line) and non-OP (thin solid line) tibiae, obtained by numbers of voxels of each HAp density normalized by the total number of voxels.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The utility of SRµCT to quantify cortical microstructure was presented here by demonstrating the difference in tibial cortical microstructure between neurectomized and contralateral intact hindlimbs of growing rats. Tibial cortical bone subjected to prolonged disuse was atrophied, and, also, its canal network was markedly reduced. On the other hand, disuse seemed to have no influence on bone mineral density. Few SRµCT as well as conventional µCT studies have focused on cortical microstructure. To our knowledge, only one group at European Synchrotron Radiation Facility in Grenoble, France has reported the SRµCT studies on cortical microstructure in mice femora (23, 24) and human femoral necks (4). In those canal-imaging studies, the resolution was lower than that in the present study, and no canal rarefaction was found in mice subjected to hindlimb unloading produced by tail suspension (24).

Advantages of SRµCT.   The major advantage of SRµCT over conventional µCT is the availability of monochromatic and high-intensity X-rays. Beer’s law, which serves as a foundation for computed tomography algorithm, holds for monochromatic X-rays but not for polychromatic X-rays used in conventional µCT. In the latter, beam-hardening artifacts, that is, an artificial darkening at the center and a corresponding brightening near the edges, are inevitable due to the preferential absorption of low energy X-rays (17). It is commonly difficult to differentiate between those artifacts and actual material variations of absorption coefficients, because beam hardening depends both on material composition and path length through the sample. On the other hand, practically no beam-hardening artifacts and a high signal-to-noise ratio are expected in SRµCT, owing to the availability of X-rays with monochromaticity but, nonetheless, extremely high light intensity. Actually, the distribution of X-ray absorption through K2HPO4 solution was almost flat (Fig. 1B).

Owing to no beam-hardening artifacts, the quantification of bone mineralization is possible in SRµCT, which is validated here by the high-linear relationship between the concentration and the X-ray absorption of K2HPO4 solution (Fig. 1C). Theoretically, the mass absorption coefficients of K2HPO4 (µ/{rho}) and water (µwater/{rho}water) are expressed using A and B in Eq. 1 as follows (33):

(3)

(4)
where {alpha} is a dimensionless constant (0.68) and {rho}water is the water density (1.0 g/cm3), linking the concentration (c) to the density ({rho}) of K2HPO4 solution by the relation:

(5)
Substitution of the experimental values of A and B yields µHAp/{rho}HAp = 5.71 cm2/g and µwater/{rho}water = 0.81 cm2/g, which matches the theoretical values given by the NIST database with precisions of <8 and <1%, respectively. In addition, the linear absorption coefficient predicted for the HAp ossicle of uniform density (3.12 g/cm3) differed by only 2.4% from the value determined by SRµCT. These results confirmed the validity of the present calibration method, translating the SRµCT image into the distribution of bone mineral density.

The absence of beam hardening will also be beneficial in obtaining metric properties of canals, such as diameters and volumes. Simple thresholding results in almost the same mineral density at every canal-bone boundary for monochromatic SRµCT images. However, in binarized polychromatic µCT images, mineral densities at canal-bone boundaries differ within or between samples, because beam hardening precludes a one-to-one correspondence between the mineral density and the linear absorption coefficient. That is, the use of monochromatic SRµCT allows determination of canal metric properties without the effects of sample thickness and canal location. Beam-hardening artifacts may not be substantial for present small samples though.

Disuse effects on growing cortical bone.   During the growth phase, more bone is deposited than resorbed, yielding a positive bone balance. Disuse or immobilization caused by sciatic neurectomy, however, decelerates bone gain and induces bone atrophy, as shown in Fig. 4. The smaller CTV and CTA and the slightly larger MA in OP tibiae imply that disuse-induced deceleration of bone formation, acceleration of bone resorption, or both occur, especially at the periosteal side. These tendencies are similar to those observed in earlier studies (47, 50). On the other hand, sciatic neurectomy had no influence on tibial bone mineral density (Fig. 5). This result is different from the finding of reduced tibial mineralization under disuse (15, 28) but consistent with earlier studies, showing no neurectomy-induced reduction in distal tibial mineralization (30, 49). Sciatic neurectomy would have little effect on mineralization in the tibial diaphyseal segment immediately above the tibio-fibula junctions.

Regarding cortical microstructure, all indexes demonstrate that cortical canals are smaller in cross section, more sparsely distributed, and less connected in OP tibiae (Table 1). These index differences imply the possibility of disuse-mediated regression of microvascularity, because its distribution is facilitated through the cortical canal network. Interestingly, the capillary network in disuse-induced atrophic soleus muscle is reduced in a similar manner as the present canal network rarefaction, i.e., decreases of capillary diameter and the number of anastomoses (12), the latter of which could correspond to the number of CaLn in the present study. Cortical microvascular rarefaction, if any, will lower bone perfusion and enhance perfusion heterogeneity (14).

The mechanism underlying disuse-mediated canal network rarefaction is beyond the scope of the present study. However, it could be speculated that loss of mechanical loading is responsible for such rarefaction during the growth phase. Bone matrix deformation or interstitial fluid flow caused by loading induces osteocyte- and vascular endothelium-derived signals (6, 26, 27, 31, 34), which would regulate bone resorption and formation around the cutting and closing cone of a progressing basic multicellular unit. Vascular growth or angiogenesis involved in basic multicellular unit progression would also be driven through those signals (7, 21, 44). During the growth phase, the coupling of cortical canalization and vascularization might be coordinated through those signals to support rapid bone gain. Therefore, loss of loading would disturb this coupling through decreasing interstitial fluid movement, which was actually observed in rat femora with hindlimb unloading (9).

Study limitations.   Although the smaller canal volume fraction and the smaller cross-sectional area were observed in OP than in non-OP tibiae, canal sizes were close to the resolution limit for both groups. Assuming a circular canal cross section and its diameter as indicative of the size of a canal, we found average canal diameters of 15.7 and 17.5 µm for OP and non-OP tibiae, respectively, which correspond to just a few voxels. Actually, the smallest visible canals had a diameter as small as the voxel size, leaving open the high possibility that the specimens contained canals too small to be picked up with the present resolution. Thus artificial canal disconnections or partial volume effects might distort the index values (Table 1), especially the canal connectivity-sensitive indexes, CaLn/CTV and CaLp/CTV. More accurate measurements can be taken by SRµCT arranged for resolving a sample into smaller voxels, if needed, down to a level of submicrometer (43, 45); however, the higher resolution necessitates the smaller view field, which is unfavorable for characterizing cortical microstructure.

The observed canal network gives a conjecture about features of cortical microvascular network; however, it is not clear whether those canals actually contain blood vessels. Some of the canal pores may be just spaces where vessels ever existed or vascularization is in progress. Furthermore, whether vascular diameters are correlated with canal diameters is not clear. A combination of SRµCT with a vascular casting method would allow the simultaneous measurements of cortical canal and vascular network structure based on the X-ray resorption difference between bone HAp and vascular contrast medium. In such measurements, however, higher spatial resolution will be required than in the present SRµCT, because some vascular canals may contain a capillary of <5 µm in diameter, as observed in atrophic muscle (12, 18). Concomitant vascular labeling will also be required to identify blood vessels within canals.

It is worthwhile to note that the intact leg will be used to compensate for the neurectomized leg by increased weight bearing. Accordingly, OP tibiae may be subjected to additional stresses, making the present tibial structural differences larger than those between neurectomized and intact rats.

In conclusion, SRµCT was shown to be useful for the cortical microstructural analysis, providing 3D information on both canal network architecture and bone mineralization. Application of SRµCT to tibial cortical bone of growing rats undergoing unilateral sciatic neurectomy demonstrated disuse-mediated canal network rarefaction, as well as bone atrophy, but no change in bone mineral density. The mechanism underlying this cortical microstructural alteration and its relevance to cortical microcirculation needs to be investigated further.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
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Part of this study was supported by Grants-in-Aid for Scientific Research on Priority Areas (15086210) and for Scientific Research B (17300152) from the Ministry of Education, Science, Sports and Culture.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The synchrotron radiation experiments were performed at the SPring-8, with the approval of the Japan Synchrotron Radiation Research Institute.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Matsumoto, Machikaneyama-cho 1-3, Toyonaka 560-8531, Japan (e-mail: matsu{at}me.es.osaka-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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