**4.2. Imaging and biological applications of bone scaffolds**

The 3-D porous structure and sufficient mechanical stiffness of the bone scaffolds are necessary conditions for the attachment, growth, and progress of mesenchymal osteoprogenitor stem cells (Prosecká et al., 2011); see Fig. 3.

**Figure 3.** Micro-CT scans of a bone scaffold manufactured from collagen/hydroxyapatite matrix. A – a single section acquired with a 4× objective; the scanning time was 9 hours. B – an image reconstruction based on 3376 sections. The scale bars indicate 1 mm.

Traditional methods for evaluating the osseointegration of tissue-engineered scaffold/cell constructs are based on 2-D histological and radiographic techniques. Sectioning followed by histology can image the scaffold interior but is destructive, lengthy and only semiquantitative (Ho & Hutmacher, 2006). Fluorescence microscopy can be quantitative when high-throughput approaches are applied, e.g., producing 3-D images with confocal fluorescence microscopy (Tjia & Moghe, 1998). Colorimetric and fluorometric soluble assays are available for cell components, such as enzymes, protein or DNA (Ho & Hutmacher, 2006). However, these soluble assays are quantitative but do not provide information on cell distribution. In contrast to these methods, by micro-CT we can non-destructively obtain 3-D images that penetrate deep into the scaffold interior and produce inherently quantitative 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 osteon to gross bone morphology.

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

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

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).
