**4. Micro-CT imaging of biological samples** *ex vivo*

### **4.1. Biomechanics of bone scaffolds**

The aim of this chapter is to illustrate the application of micro-CT in tissue engineering and in assessing the biomechanics of biocompatible collagen/hydroxyapatite bone scaffold 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 mesenchymal stem cells.

176 Injury and Skeletal Biomechanics

methods.

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

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

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

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

The aim of this chapter is to illustrate the application of micro-CT in tissue engineering and in assessing the biomechanics of biocompatible collagen/hydroxyapatite bone scaffold

pigment, and gelatin with bismuth or barium sulfate (Zagorchev et al., 2010).

walls in atherosclerosis or other pulmonary vascular diseases (Razavi, 2012).

**4. Micro-CT imaging of biological samples** *ex vivo*

**4.1. Biomechanics of bone scaffolds** 

(voxel size of 10-7 mm3) (Ritman, 2011; Müller, 2009).

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 tissue rupture, could be obtained.

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).

From a biomechanical point of view, the shape of the stress-strain curves was identical for all compositions of scaffolds. The beginning of each stress-strain curve was linear with low stiffness, and increasing loading led to scaffold stiffening. This stiffening was likely caused by the fact that, with increasing deformation of the porous specimens, the originally high amount of free space filled by air became smaller, and the stiff components became closer together and started to contribute more to the mechanical response. The decreasing porosity caused by the increasing pressure loading could thus be connected with the higher stiffness of the scaffold. The initial modulus of compression increased rapidly with an increase in the collagen concentration. From these two results, we can conclude that not only the composition but also the porosity may play crucial roles in the mechanical properties of collagen/hydroxyapatite composite bone scaffolds.

Correlating Micro-CT Imaging with Quantitative Histology 179

results (Tjia & Moghe, 1998). Micro-CT currently appears to be the most suitable approach for this task (Ho & Hutmacher, 2006; Mather et al., 2007; Mather et al., 2008; Cancedda et al., 2007). Clearly, in a structure as complex as bone, micro-CT provides a distinct advantage over conventional microscopy. Structures can be followed continuously, from the level of

Dorsey et al. (2009) explored the use of X-ray microcomputed tomography for observing cell adhesion and proliferation in polymer scaffolds. The ability of micro-CT to detect cells in scaffolds was compared with those of fluorescence microscopy and a soluble DNA assay. The researchers demonstrated that fluorescence microscopy had better resolution than micro-CT and that the soluble DNA assay was approximately 5 times more sensitive than micro-CT. However, by micro-CT, they were able to reveal the interiors of opaque scaffolds and to obtain quantitative 3-D imaging and analysis via a single, non-invasive modality. They observed that for quantitative micro-CT volume analysis, a cell density of more than 1 million cells/ml is required in the scaffolds. The results demonstrate the benefits and limitations of using micro-CT for the 3-D imaging and analysis of cell adhesion and proliferation in polymer scaffolds. Various bone engineering groups have noted the importance of micro-CT analyses in tissue engineering. Among them, Müller and Rüegsegger have investigated, and quantified, the architecture of cancellous bone using

In addition to these analyses, a number of morphological measures are being investigated, such as volume fractions of tissues, directivity of calcifying tissue, porosity, pore connectivity, putative vascularization, curvature and surface-to-volume measure (Bentley et

Despite the advantages of using micro-CT, there are still several issues with both image segmentation and resolution that are exacerbated by the low image contrast due to the low X-ray attenuation of the materials being used (Mather et al., 2008). Morris et al. (2009) studied a method for the generation of computer-simulated scaffolds that resemble foamed polymeric tissue scaffolds. They showed that the quality of the images (and hence the accuracy of any parameters derived from them) may be improved by a combination of pixel binning and by taking multiple images at each angle of rotation. However, micro-CT is considered to be a standard technique in tissue-engineered bones (Cancedda et al., 2007).

Anatomical corrosion casts provide 3-D insights into the arrangement of hollow structures and organ cavities at both the macro- and microscopic levels. The most frequently used are vascular corrosion casts (Giuvărăşteanu, 2007), which, in combination with scanning electron microscopy, constitute the primary application of corrosion casting describing the morphology and anatomical distribution of blood vessels (Lametschwandtner et al., 2004; 2005). Many other examples are found in the literature - the airways (Hojo, 1993), bile ducts

**5. Three-dimensional imaging of microvascular corrosion casts** 

micro-CT (Müller, 1994; Müller et al., 1996, 1998; Rüegsegger, 2001).

osteon to gross bone morphology.

al., 2002; Jorgensen et al., 1998).

**5.1. Corrosion casting** 
