**3. Current applications of micro-CT in biomechanics and medicine**

174 Injury and Skeletal Biomechanics

according to Roth et al., 2010).

**Figure 1.** Configuration of a micro-CT scanner with an *ex vivo* sample rotating within a stationary X-ray

More sophisticated devices use microscope objectives for increased magnification and resolution. For optimal settings of the micro-CT scan, the spot size (SS) and the awaited pixel size (PS) are the parameters that set the source distance (SD) and detector distance (DD). When the SD and DD are smaller, higher photon counts can be achieved, thus

*DD PS SS*

**Figure 2.** For optimal settings of the micro-CT scan, the spot size (SS) and the awaited pixel size (PS) are the parameters that set the source distance (SD) and detector distance (DD) (redrawn and modified

The source SS is strongly dependent on the power of the X-ray tube used. In today's machines, the source SS ranges between 1-10 µm. For high resolution scanning of biological samples, it is advisable to operate with a low accelerating voltage (the typical range for the Xradia XCT 400 (Xradia, Pleasanton, CA, USA) is 20–60 kV used with a power of 4 W, at which the tube yields the lowest SS). The scanning time depends on the magnification and resolution required. For

*SD*

(1)

reducing the time costs. The settings should fulfill equation 1; see also Fig. 2:

system (redrawn and modified according to Jorgensen et al., 1998).

Compared with standard human CT devices, which offer a resolution limit of approximately 0.4 mm, the micro-CT introduced a promising modality. However, the clinical use of this method is limited by its higher radiation exposure and longer scanning times. It is used either to visualize individual fine functional and anatomical structures of *ex vivo* human or animal organs (e.g., liver lobules or bone trabeculae) or for whole-body imaging of small animals (Schambach et al., 2010). *In vivo* micro-CT systems are based on a rotating system of X-ray tube and detectors. The construction of these devices is the same as in human CT, except that their dimensions are adapted to small animals (Bag, 2010). The minimal space resolution of *in vivo* micro-CT is from 100 to 30 µm. The imaging of living animals must be faster than in *ex vivo* micro-CT. It is enabled by, among other factors, the use of flat panel detectors that allow us to acquire an abundance of thin sections during one rotation. The examination is also limited by the necessity of using a radiation dose that does not harm the tested animal. In *ex vivo* micro-CT, the X-ray source and the detectors are stationary, and it is possible to adjust the distance between the X-ray tube and the detectors (based on the size of the examined object) to improve the spatial resolution and minimize artifacts. Moreover, in *ex-vivo* micro-CT, time resolution is not important; thus, the examination may take a very long time (hours), and any amount of radiation may be used. This technique enables us to acquire much higher spatial resolution than *in vivo* micro-CT (30 to 1 µm) (Zagorchev et al., 2010).

Most organs have already been analyzed with micro-CT, including bones (Peyrin, 2011), heart and blood vessels (Schambach et al., 2010), lungs, kidney, liver, and cerebral structures (Schambach et al., 2010). Micro-CT devices can be used for the characterization of bone or vascular microarchitecture (Peyrin, 2011; Burghardt et al., 2011; Missbach-Guentner et al., 2011). This method also allows the precise detection of the margins of tumors and their vascularity (Ma et al., 2011; Missbach-Guentner et al., 2011). Tissue composition (e.g., bone mineralization) can be directly linked to 3-D tissue morphometry (Burghardt et al., 2011). Thus, 3-D micro-CT analysis becomes a method of choice for describing the spatial complexity of organ segmentation and the relationships between morphological and functional units (e.g., hepatic lobules and portal acini) (Schladitz, 2011). Micro-CT can also link the imaging of anatomical structures with functional and molecular imaging, e.g., tissue and organ perfusion, the flow rate of exocrine secretions within parenchymatous organs and glands (Marsen et al., 2006), or heart movements (Badea et al., 2005).

For the examination of soft tissues and vessels, contrast solutions are necessary. In *in vivo* imaging, such as in human medicine, standard iodinated contrast media or intravascular blood-pool contrast agents are used (e.g., contrast material covered by a polyethylene glycol capsule and stabilized by lipoproteins or iodinated triacylglycerides). These blood-pool contrast media are able to remain in the blood circulation for a longer time and thus enable longer scanning times. They do not leave the blood circulation as do standard iodinated contrast agents, which pass into the extravascular interstitial space. Contrast substances labeled with antigens or other ligands (Ritman, 2011) may also be used for targeting and tracking specific structures, such as stem cells (Villa et al., 2010). For *in vivo* studies, nanoparticles can be used to enhance the soft tissue contrast (Boll et al., 2011). It is also possible to use nanoparticles that incorporate into, e.g., tumors and could remain there for longer periods (Boll et al., 2011). However, *ex vivo* micro-CT can utilize any contrast solution, including those that are toxic to living organisms. The only limitation is that they must not damage the examined tissue. In *ex vivo* micro-CT imaging, it is recommended that contrast solutions be used that offer high contrast to the studied tissue, that have a low viscosity to fill the smallest vessels and that do not diffuse out of the blood vessels. In practice, the substances used include silicon rubber (Savai et al., 2009), polymethylmethacrylate with added lead pigment, and gelatin with bismuth or barium sulfate (Zagorchev et al., 2010).

Correlating Micro-CT Imaging with Quantitative Histology 177

samples (Prosecká et al., 2011). Tissue engineering is a promising interdisciplinary research field that aims to develop biological substitutes for the repair of damaged tissue. The typical strategy involves either the delivery of isolated and expanded cell populations within a tissue engineering construct or the recruitment of host cells local to the site of damage through the use of conductive scaffolds and inductive biological signals. The question of how to optimize the design of scaffolds for different tissues remains unsolved. To assess the suitability of polymer tissue scaffolds for use in regenerative medicine, methods to characterize scaffolds are needed (Renghini et al., 2009). The bone scaffolds should be stiff enough to withstand high forces in the bone after implantation but, conversely, should also be flexible enough to enable growth of the cells and changing of the original shape of the graft to meet the needs of the bone complex. Therefore, it is important to perform mechanical measurements and 3-D imaging of bone scaffolds before the seeding of

Generally, bones and bone scaffolds can be mechanically tested using various types of techniques: tensile or torsion tests used for strip- or block-shaped tissue specimens; a pressure test used for block-shaped or cylindrical specimens; a ring test in which a ring of given thickness is cut from a tubular organ (typically a blood vessel), clamped into the jaws of special measurement devices and loaded by tension; and an intraluminal pressure inflation-deflation test of tubular organs. The choice depends on the physiological loading of the tissues. The aim is to be as close as possible to the real loading and thus to the real mechanical properties. In any case, regardless of the chosen technique, the result given by a measurement device is of the same nature: a stress-strain (tensile, pressure, ring test) or pressure-outer diameter (inflation-deflation test) curve. The stress-strain curve has a mostly nonlinear shape showing the tissue stiffening as loading increases. The stiffening is caused by various tissue components as they subsequently contribute to the tissue response. The soft component, mostly elastin, contributes to the mechanical response at low loading and is connected with the low stiffness of the tissue, whereas the curly and stiff collagen fibers are straightened as loading progresses and contribute to stiffening at high loading. The relevant portions of a stress-strain curve could be approximated (e.g., by a line), and thus, the mechanical parameters, such as Young's modulus of elasticity at small deformations (low loading) and at large deformations (high loading), the pressure-strain elastic modulus, the initial modulus of compression, the limit stress and the strain in the case of loading until

To emulate the loading of bones, in which most of their parts are under pressure and only a small fraction of the tissue is under tension, pressure loading was applied to collagen/ hydroxyapatite composite bone scaffolds. Cylindrical specimens (approximately 12 mm in diameter and in height) of composite scaffolds containing various amounts of collagen and hydroxyapatite prepared according to Prosecká et al. (2011) underwent pressure mechanical loading with a loading velocity of 1 mm/min. The resultant stress-strain curves, and particularly the regions between 2% and 10% of the original specimen's height, were approximated by linear regression, and thus the initial moduli of compression were determined. (for details of the measurements and their evaluation, see Prosecká et al., 2011).

mesenchymal stem cells.

tissue rupture, could be obtained.

Other modifications of CT applicable in medical experiments are the mini-CT devices (voxel size 10-3 mm3, used for scans of whole organs or small animals) and the nano-CT devices (voxel size of 10-7 mm3) (Ritman, 2011; Müller, 2009).

The greatest progress in micro-CT exploration was acquired in the exploration of tumor microvascularization and the study of neoangiogenesis. The latter phenomenon is also important for the understanding of tumor growth and could be used in oncological treatment strategies, especially in patients treated by biological therapy with antibodies against vascular endothelial factor A (bevacizumab), which inhibits neoangiogenesis (Ma, 2011). Micro-CT imaging of pathological vascularity can provide new information, e.g., about changes in vessel walls in atherosclerosis or other pulmonary vascular diseases (Razavi, 2012).

A promising trend in experimental work is using hybrid methods that combine detailed anatomical information from micro-CT with information about cellular metabolism and structure from methods of nuclear medicine (micro-SPECT/CT and micro-PET/CT) (Ritman, 2011). Therefore, it is appropriate to combine or compare the results of micro-SPECT or micro-PET with, e.g., microscopic analysis of a specimen to estimate the correct anatomical orientation and acquire a satisfactory interpretation of the results. The new suggested tools would be able to use higher energy examination from more X ray sources and thus obtain results on the cellular or subcellular level. The development of new tissue-specific contrast solutions could also be promising for future research activities using micro-CT or its hybrid methods.
