Curr Osteoporos Rep

DOI 10.1007/s11914-014-0233-0

IMAGING (T LANG AND F WEHRLI, SECTION EDITORS)

Micro- and Nano-CT for the Study of Bone Ultrastructure

Françoise Peyrin & Pei Dong & Alexandra Pacureanu &
Max Langer

# Springer Science+Business Media New York 2014

Abstract Micro-computed tomography (micro-CT)—a ver- Introduction

sion of X-ray CT operating at high spatial resolution—has
had a considerable success for the investigation of trabecular Bone fragility diseases such as osteoporosis are still the focus
bone micro-architecture. Currently, there is a lot of interest in of active research to understand the mechanisms involved in
exploiting CT techniques at even higher spatial resolutions to bone loss and in bone failure. Although bone mass is an
assess bone tissue at the cellular scale. After recalling the basic important determinant of bone strength, it is known not to be
principles of micro-CT, we review the different existing system, sole factor. According to Wolff’s law, it has the property to
based on either standard X-ray tubes or synchrotron sources. adapt to the mechanical constraints to which it is subjected.
Then, we present recent applications of micro- and nano-CT for Bone fragility is hypothesized to be the result of failed mate-
the analysis of osteocyte lacunae and the lacunar-canalicular rial or structural adaptations to mechanical stress [1, 2]. Bone
network. We also address the question of the quantification of adaptation results both from the bone modeling and remodel-
bone ultrastructure to go beyond the sole visualization. ing processes. Thus, in response to loading, bone is expected
to change its macro, micro, and nano structure.
Bone remodeling is achieved via mechanotransduction, a
Keywords Computerized tomography . Synchrotron process in which the osteocyte system plays a major role
imaging . Micro-CT . Nano-CT . X-ray phase CT . Bone [3–6]. Osteocytes are cells deeply buried in bone tissue com-
ultrastructure . Osteocyte lacunae . Canaliculi . Lacunar municating with each other through dendritic processes. Os-
canalicular network . Bone quality . Lacunar density . teocytes and their processes are encapsulated within the so-
Quantification called lacunar-canalicular network (LCN) [7]. Interstitial fluid
flows circulating in the LCN are hypothesized to stimulate
osteocytes. Microcracks could also interrupt cellular processes
F. Peyrin : P. Dong : M. Langer
and participate in the transduction to trigger the repair process
INSERM U1044, CREATIS, 69621 Villeurbanne, France
[8, 9]. For several years there has been a phenomenal interest
F. Peyrin : P. Dong : M. Langer in these bone cells qualified with expressions such as “the
INSA-Lyon, 69621 Villeurbanne, France unrecognized side of bone tissue” or “can’t hide forever”
F. Peyrin : P. Dong : M. Langer
[10–14]. In addition to their participation in osteoclast recruit-
CNRS UMR 5220, 69621 Villeurbanne, France ment and the initiation of bone remodeling, they have a role in
maintaining mineral homeostasis. They secrete a number of
F. Peyrin : P. Dong : M. Langer factors, among which some are seen as potential therapeutic
Université de Lyon, 69622 Lyon, France
targets [15].
F. Peyrin : P. Dong : M. Langer However, little quantitative data is available on the com-
Université Lyon 1, 69622 Villeurbanne, France plex organization of the osteocyte network within osteons and
interstitial tissue. This lack of data can be attributed to both a
F. Peyrin (*) : P. Dong : A. Pacureanu : M. Langer
long-time lack of interest in the osteocyte system but also to
European Synchrotron Radiation Facility, BP 220, 38043 Grenoble,
Cedex, France the limited means for investigating this structure. Imaging the
e-mail: [email protected] osteocyte system of the LCN is quite challenging because of
 Curr Osteoporos Rep

its interior location within bone matrix, the small size of In contrast to light, X-rays easily penetrate through bone
lacunae (a few micrometers) and canaliculi (100–700 nm in samples and X-ray imaging is particularly well suited for the
diameter), and the complexity of this network. The LCN analysis of bone at different scales [33]. At the micro structure
forms a very dense network with several thousands of lacunae scale, X-ray micro-CT has had considerable success for the
per mm3 and hundreds of thousands of canaliculi per mm3 analysis of trabecular bone, since it provides, nondestructive-
constituting a tight meshwork within bone tissue. ly, a very large number of histology-like slices and direct 3D
Much knowledge on the osteocyte system comes from the quantitative parameters of cancellous bone. It is also a tech-
early work of Marotti et al. [16, 17]. In 1979, these authors nique of choice to investigate the Haversian and Volkmann
already characterized the three axis of the ellipsoidal shape of porosity in cortical bone [34, 35]. While micro-CT systems
the osteocyte lacunae by using bone slices in different direc- used to image bone micro-structure are not directly adapted to
tions and determined that the major axis was parallel to the investigate bone ultrastructure, nano-CT systems have been
main orientation of the collagen fibers [18]. They later showed built for that purpose. Nevertheless, reaching the appropriate
that osteocytes were located within loose cellular lamellae in spatial resolution is a key issue to image ultrastructural
comparison with dense acellular lamellae [19]. features.
In most works, microscopic imaging techniques using either In this paper, we review how recent developments in X-ray
visible light, Scanning Electron Microscopy (SEM) or Trans- CT systems are pushing CT imaging toward the cellular scale
mission Electron Microscopy (TEM) have been used, the latter and beyond. In Section Two, after recalling the basis of X-ray
achieving the images at the highest spatial resolution. Neverthe- CT, we introduce the advances in micro/nano-CT technology
less, most of these techniques provide only two-dimensional using either conventional X-ray tubes or X-rays extracted
images making it difficult to obtain a complete understanding of from synchrotron sources. In Section Three, we review the
the complex network. Several studies used stereology to extrap- recent use of these systems to image the lacunar-canalicular
olate two-dimensional (2D) measurements to three-dimensional network as well as properties of the bone extra cellular matrix.
morphologic parameters [20]. However, such extrapolations are
based on ideal shape models and can be biased due to inappro-
priate assumptions or to variations in the slicing direction. Thus, CT Techniques
ideally the LCN should be investigated with a three-dimensional
(3D) imaging modality providing sufficiently high spatial reso- From X-Ray CT to X-Ray Nano-CT
lution. This was the conclusion of review paper from Schneider
et al, which detailed the existing techniques and pointed out their The generic principle of X-ray CT is based on the combination
limitations [21••]. of data acquisition and data processing to produce 2D images
Three-dimensional imaging of bone ultrastructure is cur- of the anatomy. Data acquisition is performed by rotating an
rently a hot topic of research [22]. Confocal Laser Scanning X-ray source around the patient or sample and measuring the
Microscopy (CLSM) in which serial 2D optical microscopy attenuation of X-rays as they pass through the body. The
images are acquired at consecutive layers by scanning the measures of attenuation do not directly provide the image,
sample through the microscope focus has been the most but a so-called sinogram. Then, by using a 2D tomographic
widely used technique. It can reach high spatial resolution reconstruction algorithm such as Filtered Back Projection
typically around 200 nm in the focal plane and approximately (FBP), relying on an exact mathematical relationship relating
450 nm in depth. Moreover, it can be coupled to a variety of the image to its sinogram, the CT image is obtained.
fluorescence labels permitting to stain different cells [23]. This Clinical CT is used daily to image bone at the organ level
technique has, for instance, been used to obtain three- but does not offer a sufficient spatial resolution to reveal bone
dimensional renderings of the osteocyte network in bone of microstructure. Since a gain in spatial resolution in CT is
animals [24, 25] and of women with and without fractures achieved at the cost of an increase of radiation exposure, this
[26]. A number of recent studies investigating various prop- limits the possibility for in vivo imaging. High-resolution
erties of the osteocyte network are based on CLSM [27–29, pQCT systems can now achieve spatial resolution as high as
30•, 31]. Although the technique shows a potential, it still has 150 micrometers but are restricted to peripheral bone (radius,
some limitations, which includes decreased of contrast with tibia) [36].
depth and a maximum depth of about 100–150 μm due to the It is technically possible to reach much higher spatial
limited penetration and diffusion of visible light within hard resolution for ex-vivo imaging. Experimental setups and com-
tissues such as bone. In addition, the lower spatial resolution mercial X-ray micro-CTs have been developed to image bone
in the depth direction makes the images more difficult to micro architecture [37]. Most systems use a cone beam X-Ray
analyze qualitatively and quantitatively. Other techniques source associated with a two-dimensional detector. When the
such as coupling Focused Ion Beam (FIB) and SEM have X-ray source is circularly rotated around the sample, a set of
been proposed but they have remained exploratory [32]. 2D radiographs at different view angles are recorded. The
Curr Osteoporos Rep

detector may be composed of a scintillator converting X-ray for the simultaneous assessment of structure and mineraliza-
photons to light coupled to a 2D detector. The 3D image is tion in human or animal trabecular bone [42, 43]. Its applica-
generally reconstructed using the Feldkamp-David-Kress tion to cortical bone also permits quantification of the
(FDK) algorithm, which generalizes the FBP algorithm [38]. Haversian canal network and visualize osteons [34, 44, 45].
This method is not exact as in the 2D case and can produce so- SR CT implemented at various synchrotron sources has
called cone beam artifacts. The errors increase with the diver- been used at multiple spatial resolutions, ranging from 10 to
gence of the cone beam but remain low for cone angles 0.7 μm. The system developed on beamline ID19 at the ESRF
smaller than 10°. They are more important in the top and is based on 3D parallel beam tomography and can provide
bottom slices, which are sometimes excluded from the images at nominal voxel size up to 0.2 μm [46]. The X-ray
reconstruction. beam energy can typically be chosen in the range from 12 to
Different machines with different characteristics in terms of 80 keV by using a double crystal monochromator. The 3D
X-ray voltage, current, and spatial resolution are available image is reconstructed from 1000 to 2000 radiographs, by the
since it is not possible for a single micro-CT system to cover FBP algorithm performed on each slice. Setting a configura-
all ranges of spatial resolutions between 100 μm to 1 μm. The tion at a given voxel size requires a number of adaptations in
choice of spatial resolution is a key issue to obtain a satisfac- the beam size and flux as well as in the detection part, ie, the
tory image of a given structure. Trabecular bone is generally scintillator and eventually CCD detector.
imaged with voxel size between 5 μm and 20 μm, correspond- Synchrotron sources also give access to phase contrast
ing to fields of view of several centimeters (depending on the imaging. Propagation-based X-ray imaging is the simplest
number of pixels of the detector). Imaging lacunae and cana- experimental implementation of phase contrast imaging and
liculi requires spatial resolutions at the micrometer or the can be coupled to CT [47]. Acquisition of a phase CT image
nanometer scale. consists of recording one or several scans at different detector
A new generation of machines called X-Ray nano-CT has distances. The reconstruction algorithm includes a phase re-
more recently been optimized to achieve ultra-high spatial trieval step prior to FBP reconstruction [48]. This technique
resolutions. They generally use a nano focal spot source provides a map of the so-called phase index decrement (δ),
(<400 nm). Various vendors currently offer such machines. which is related to the electron density and yields higher
As an example, Bruker now offers a multi-scale X-ray nano- sensitivity compared with the linear attenuation coefficient
CT system (SkyScan2211) based on a cone beam design using obtained in absorption CT.
an open pumped X-ray source with a voltage between 10 and Phase contrast imaging has been exploited to develop a
190 kV and a 14-bit detector (6 M pixels flat panel or magnified X-ray Nano-CT setup at the nano-imaging station
11 M pixels cooled CCD). The spatial resolution given by ID22NI of the ESRF [49]. The beam is magnified by using a
the vendor is 600 nm at 10 % modulation transfer function. Kirkpatrick-Baez optical system. The X-ray energy can be set
However, due to the limited flux, the signal-to-noise ratio between 17 keV and 30 keV. Typically three or four scans of
(SNR) may be low. The system can scan objects between the sample recorded at different distances from the focal spot
1 mm for the higher spatial resolutions (voxel size: 0.1 μm) of the source are recorded. The system provides images with
up to 204 mm for lower spatial resolutions. isotropic voxel sizes between 25 and 400 nm.
It is worth noting that the radiation exposure increases with
From Synchrotron X-Ray Micro-CT to Nano-CT the image spatial resolution and can reach 1 or several MGy
[50, 51••]. Thus, if bone samples are analyzed with various
In parallel to desktop X-ray micro-CT, synchrotron radiation techniques, very high spatial resolution X-ray imaging should
CT setups have also been developed to investigate bone micro be kept as the last step since it has an impact on bone
architecture [39, 40]. Using synchrotron sources presents ad- mechanical properties [52].
vantages for CT, especially for imaging samples up to the
nanometer scale. Synchrotron sources offer a photon flux
several orders of magnitude higher than that of conventional
X-ray tubes. This property becomes particularly important for X-Ray CT Imaging of Osteocyte Lacunae
sub-micrometer spatial resolution imaging, thereby limiting
scan times while achieving high SNR. They also permit use of Standard micro-CT systems used for the investigation of tra-
monochromatic X-ray beams, avoiding the beam-hardening becular architecture have been reported to be limited for the
artifacts. The resulting image can then be considered as a map visualization of osteocyte lacunae [53]. This was predictable
of the linear attenuation coefficient within the sample. Since since imaging osteocyte lacunae requires a spatial resolution
this coefficient is related to the composition of the sample, it better than 5 or 10 μm. Two initial studies have reported the use
was used to estimate the degree of mineralization of bone in of X-ray nano-CT to examine the 3D morphology of osteocyte
3D [41]. During the past decade, SR micro-CT has been used lacunae [54, 55]. In the first study, the authors analyzed bone
 Curr Osteoporos Rep

samples from the calvaria and fibulae in adult mice by using more elongated and flattened lacunae in the anterior and poste-
both CLSM and nano-CT at voxel sizes of 390 nm (fibula) and rior regions, but no significant differences in lacunar volume.
480 nm (calvaria) using the Skyscan 2011 system. The nano- The same imaging technique was used to analyze the femoral
CT images revealed that the lacunae at the two sites had cortical bone in 30 women between 20 and 86 years [64••].
different morphologies and were more elongated in fibula. In While there was no variation in lacunar density a significant
the second study, the authors analyzed cortical bone samples reduction of lacunar volume was observed with age. In a recent
from the proximal tibia of three female patients with osteoar- study, we used SR micro-CT at 1.4 μm to analyze the statistical
thritis, osteopenia, and osteopetrosis. They measured lacunar properties of lacunae in human femoral bone [65•]. The bone
density, volume, surface area, and anisotropy ratio from nano- volume fraction was found to be significantly correlated to the
CT images (voxel size 580 nm) and observed significant dif- lacunar density and other lacunar features.
ferences in morphology. These two studies support the hypoth- To compare the results from various studies, it is important
esis that osteocyte lacunar morphology is affected by matrix to know which parameters are extracted from the images and
strain due to different loading conditions but these studies how they are calculated. After segmentation, counting the
remain limited in terms of numbers of samples and of analyzed number of lacunae (Lc.N) and then assessing the lacunar
lacunae. With the exception of these two studies, we are not density (Lc.N/TV or Lc.N/BV expressed in mm-3) is straight-
aware of other work using desktop nano-CT for the analysis of forward. To assess lacunar shape, it is appropriate to use an
lacunae. The limitations to diffusion of this technique could be ellipsoidal fit, which can be obtained by using the matrix of
related to the long image acquisition times and the lack of second order moments. Its 3 eigenvalues are related to axis
dedicated image analysis software to analyze the data. lengths and its 3 eigenvectors provide the three ellipsoid axes,
At the same time, while the potential of SR CT for imaging and thus, the orientation of the lacuna. Most work reported
bone at the cellular scale had been recognized early [56, 57], only the eigenvalues, which must not be confused with the
its use for the analysis of osteocyte lacunae in animal models lengths since the latter are actually proportional to the square
or in humans has considerably increased during the last few root of the eigenvalues scaled by a factor 2√5 [26, 65•]. To
years. SR micro-CT at SLS (Swiss Light Source) with a voxel avoid confusion in units, it is recommended to provide the
size of 0.7 μm was first used to assess ultrastructural proper- axis lengths directly. The lacunar ellipsoidal shape has been
ties of cortical bone in two inbred strains of mice [58]. Re- further characterized by various parameters involving ratios of
cently, the same technique was used to analyze cortical bone eigenvalues or length axis. Since all these calculations are
porosity in a mouse model of osteogenesis imperfecta [59]. performed on each lacuna, the computing time may be an
Among other differences, the authors found a higher lacunar issue to process more than 10000 lacunae per image. An
density in osteogenesis imperfecta compared with a wild type. efficient calculation method allowing additional parameters
The cortical micro-porosities in rats treated with PTH or such as the surface area, Euler number, and mean curvature
Alendronate were quantified using SR micro-CT at APS (Ad- was presented in [65•]. Such quantification provides not only
vance Photon Source) with a voxel size of 0.75 μm [60]. The the mean and standard deviation of all parameters but also its
lacunar porosity varied with treatment and was dependent on distribution within the examined ROI. Hannah et al reported a
the location of the analyzed region within the cortex (endos- bimodal distribution in the lacunar volume in their initial
teal, intracortical, periosteal). The lacunar properties in the study, but this observation was not confirmed in other work
tibial diaphysis in a control and immobilized model of rats [62]. Very recently, a complete framework was described to
were studied using SR micro-CT at Canadian Light Source extract even more parameters from such images [66••]. In the
with a voxel size of 2 μm [61]. The data revealed a significant future, the comparison of results will certainly be simplified if
decrease in lacunar density and lacunar volume in the the nomenclature and definition of parameters used to char-
immobilized group, demonstrating the effect of unloading on acterize lacunae is standardized.
the lacunar properties. The different studies as well as lacunar Figure 1 shows SR CT images acquired at three different
densities and volumes are summarized in Table 1. spatial resolutions. Figure 1a shows a region of interest in a
SR micro-CT also allowed the characterization of osteocyte slice in a human femoral cortical bone sample (voxel size
lacunae in human cortical bone (Table 1). One sample from the 1.4 μm), Fig. 1b illustrates the osteocyte lacunae after seg-
femoral shaft of a 20 year old subject was imaged at the mentation, and Fig. 1c shows a colored map of the lacunae,
Advanced Photon Source, Argonne, IL, with a voxel size of coding the ratio of the two largest lengths of the lacunae.
1.47 μm [62]. The authors analyzed the variations in lacunar
density and volume with the distance from the Haversian canal
in 11 osteons within a single image. Subsequently, the same X-Ray CT Imaging of Canaliculi
group analyzed various samples taken in anterior, posterior,
medial, and lateral regions within the same subject [63]. They X-ray CT imaging of canaliculi making the junction between
reported variations in lacunar density depending on the site, osteocyte lacunae is challenging due to the small diameter of
 Curr Osteoporos Rep

Table 1 Measurements of osteocyte lacunae parameters from desktop and SR nano-CT techniques. The number of samples, lacunar density, and average lacunar volumes are reported

Reference Imaging tech Location Groups Voxel size Sample number N.Lc (#) Density type Density (mm-3) Volume (μm3)

Vatsa et al, 2008 Desktop nano-CT Mice Fibular, Calvarial 390 nm, 480 nm 2 1790 993 - - -
van Hove et al, 2009 Desktop Women proximal tibial Osteoarthritis 580 nm 2 659 N.Lc/BV 21800 51.2
Desktop nano-CT Osteopenia " 2 120.5 N.Lc/BV 8000 179.1
Osteopetrosis " 2 458.5 N.Lc/BV 15600 97.6
Britz et al, 2012 SR μCT Rat tibia diaphysis Control 2 μm 6 - N.Lc/BV 63138 284
Immobilized " 6 - N.Lc/BV 49641 209
Hannah et al, 2010 SR μCT Man femoral diaphysis 1.47 μm 1 9807 N.Lc/TV [40000–90000] 290
Carter et al, 2013 SR μCT Man femoral diaphysis 1.47 μm 13 - N.Lc/BV [26343–37521] [378–409]
(A,P,M,L)
Carter et al, 2013 SR μCT Woman femoral diaphysis 1.47 μm 30 - N.Lc/BV 23942 252
(various ages)
Dong et al, 2014 SR μCT Human femoral mid-diaphysis 1.47 μm 13 12791 N.Lc/BV 20573 409.5
Mader et al, 2013 SR μCT Mouse femoral mid-diaphysis B6lit/lit, B6lit/+ 1.4 μm 6 26063, 32514 N.Lc/BV 44800, 38500 317, 469
C3Hlit/lit, C3Hlit/+ " 11 33799, 53287 N.Lc/BV 35700, 39000 378, 577
Schneider et al, 2007 SR μCT Mouse femoral mid-diaphysis B6-lit/lit 700 nm 1 - N.Lc/TV 65865 200
C3.B6-lit/lit " 1 - 49879 269
Tommasini et al, 2012 SR μCT Rat femoral diaphysis Control 750 nm 6 - N.Lc/TV 56470 266.0
OVX, ALN, PTH " 18 - N.Lc/TV [59510–63810] [237.0–268.1]
Carriero et al, 2014 SR μCT Mouse tibial and humeral WT 700 nm 7 6471 N.Lc/TV 72981 375
mid-diaphysis Oim " 6 12347 N.Lc/TV 127365 363
 Curr Osteoporos Rep

Fig. 1 Illustration of SR CT at different scales in human femoral bone of the lacunar-canalicular network around the top osteon of Fig. 1 (d)
samples: first row: voxel size 1.4 μm, (a) ROI in the original CT slice (lacunae in yellow, canaliculi in blue), (f) 3D display of 5 lacunae and
showing the Haversian porosity (black), the osteons (gray), the interstitial their canaliculi segmented with the minimal path method presented in
tissue (light gray), and osteocyte lacunae visible as small black dots, (b) [69]: third row: voxel size 60 nm, (g) phase nano-CT slice showing a
3D rendering of the osteocyte lacunae (yellow), (c) 3D rendering of the cement line (white) lacunae and canaliculi in black, (h) 3D rendering of
ratio of the two largest lengths of the osteocyte lacunae showing their the whole 3D image (lacunae in orange) where the collagen fibers form an
anisotropy: second row: voxel size 300 nm, (d) ROI in the original CT apparent texture, (i) detail on one lacunae and its canaliculi
slice showing osteons, osteocyte lacunae, and canaliculi, (e) 3D rendering

these structures. Up to now, standard X-ray source nano-CT The visualization of the LCN was demonstrated by using
has systems have not been successful to show these structures. the parallel beam SR micro-CT setup at ESRF with a voxel
While SR micro-CT has been reported to be a good candidate size of 300 nm [51••]. This was only possible after optimiza-
for the examination of canaliculi [21••], several works devoted tion of the detector efficiency by coupling an adequate scin-
to the analysis of lacunae using SR micro-CT outlined that the tillator and a CCD camera to minimize the dose on the sample,
canaliculi were not visible even at submicrometer resolution responsible for cracks and motion artifacts. The advantage of
[60]. this technique is that it provides 3D images of the LCN on
Curr Osteoporos Rep

FOVs covering several osteons. However, since the pixel size ray micro-CT for the quantification of bone microstructure,
was close to the canaliculi diameter, their segmentations was progress in X-ray nano-CT is expected. However, because of
challenging and more sophisticated methods than simple stringent requirements on X-ray flux at nanoscale, currently
thresholding had to be considered [67–69]. Figure 1d illus- most studies at the ultrastructural level have been achieved
trates a ROI in a raw SR CT slice (voxel size: 300 nm) and using synchrotron sources CT setups. These systems can offer
Fig. 1e a 3D rendering of the LCN in an osteon. Figure 1f spatial resolution up to a few tens of nanometers with high
shows the segmentation of the LCN using a new method SNR and fast scanning times. The feasibility of observing the
based on a minimal path approach preserving the continuity 3D morphology of osteocyte lacunae, canaliculi, and even
of the canaliculi [69]. collagen fibers has been demonstrated.
A few images of the LCN at spatial resolutions between 40 After image acquisition, quantitative data have to be ex-
and 60 nm were obtained based on experimental synchrotron tracted from the images. Recently, automated methods have
techniques. In [70], the authors used rotated transmission X-ray been proposed to calculate a number of 3D lacunae features on
microscopic images to obtain a 3D reconstruction of bone tissue large data sets (more than 10,000 lacunae/sample). However,
at 40 nm, but the technique was limited to very small FOV (15– methods to quantify the complex canalicular networks have
30 μm). The feasibility of visualizing one osteocyte lacunae yet to be developed. In addition, it is worth noting that in-
surrounded by its canaliculi (spatial resolution 60 nm) was creasing spatial resolution puts a greater demand on data
demonstrated by using ptychography, a scanning technique reconstruction and processing algorithms to analyze samples
consisting of recording a sequence of overlapping diffraction with meaningful field of view, given that the data size scales as
patterns [71]. However, the long scan times and small FOVs the cube of sample size in on dimension.
restrict applications. Recently, a Talbot interferometer for phase Irrespective of the structural features to be quantified, the
CT imaging and edge-enhanced absorption CT were combined quality of the results strongly depends on image binarization.
at SPring-8 in Japan to image the LCN in mouse bone [72]. This problem is generally ignored and image segmentation
Nevertheless, the complex acquisition procedure and long ac- performed by simple thresholding. This approach is often
quisition time hampers the practicality of the approach. inadequate when the signal-to-noise ratio is low or when one
Our group used magnified phase nano CT at the ESRF to dimension of the structure to be quantified is at the limit of the
image the LCN in 3D at a voxel size of 60 nm [73••]. spatial resolution, as for instance for canaliculi. Thus, dedicat-
Compared with previous techniques, the FOV is larger ed image segmentation methods have to be developed and
(120 μm in each direction), which includes several lacunae. evaluated, which is a major challenge since the ground truth
It provides details on the canaliculi organization and its may not be known.
branching structure but it also allows mapping of the density To obtain information beyond structure, SR CT imaging
of the mineral tissue. The notable finding was that the collagen also permits quantification of the density of the sample. At the
fibrils were visible as a textured pattern, the orientation of microscale, this property has been exploited to map the degree
which was later analyzed [74••]. As an illustration, Fig. 1g of mineralization of bone and to discriminate osteons from
illustrates a raw slice and Fig. 1h a 3D rendering of the whole interstitial tissue from absorption or phase imaging. At the
sample, where the apparent orientation of collagen fibers is ultrastructural level, phase nano-CT opens many perspectives
visible. Figure 1i shows a detailed rendering of a lacuna and its to study peri-lacunar and peri-canalicular density and to ob-
canaliculi. The same 3D phase nano-CT setup was later used serve the collagen fiber texture.
to analyze the properties of the LCN in jaw bone samples There are also some limitations to X-ray micro/nano
arising from bisphosphonate-treated patients suffering CT techniques. First, so far, this technique only permits to
osteonecrosis of the jaw [75]. visualize the pores rather than the cells or the osteocytic
Nano-CT, thus, permits a better assessment of bone tissue dendrites themselves. So the number of osteocyte lacunae
properties on the lamellar scale, including nanoporosity, quan- can overestimate the number of osteocytes. However,
tification of mineralization and collagen fibers. Due to the many studies suggest that the analysis of osteocyte lacunae
recent findings at the lamellar length scale [76–78], this is a good surrogate of the osteocytes themselves provided
should open many perspectives for the understanding of bone that the percentage of empty lacunae remains approximate-
fragility. The use of such images in modeling is also a prom- ly constant. Second, the accessibility of synchrotron
ising area [79, 80]. sources is limited.
Nevertheless, X-ray nano-CT may provide reliable and
innovative information on bone nano porosities, bone mineral,
Conclusions and extra cellular matrix at the cellular scale, which may be
crucial to learn about the pathophysiological properties of
The need for a three-dimensional assessment of bone ultra- bone tissue and more generally to gain a better understanding
structure has become more obvious. After the success of X- of bone mechanical properties.
 Curr Osteoporos Rep

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Human and Animal Rights and Informed Consent All studies by the
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2010;47:848–58. Highlights the need and possible solutions for
approval by the appropriate institutional review boards. When required,
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written informed consent was obtained from all participants.
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