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

### **5.1. Corrosion casting**

178 Injury and Skeletal Biomechanics

collagen/hydroxyapatite composite bone scaffolds.

based on 3376 sections. The scale bars indicate 1 mm.

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

osteoprogenitor stem cells (Prosecká et al., 2011); see Fig. 3.

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

The 3-D porous structure and sufficient mechanical stiffness of the bone scaffolds are necessary conditions for the attachment, growth, and progress of mesenchymal

**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

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

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 (Gadžijev & Ravnik, 1996), urinary tract (Marques-Sampaio et al., 2007) and lymphatic system (Fujisaka et al., 1996) have been visualized by means of corrosion casting. The steps for obtaining the corrosion casts are as follows: perfusion of the hollow target structures to remove the contents (in the case of vascular casts, prior heparinization is required for the maintenance of blood fluidity), injection of the casting media, its polymerization in the fully filled cavities and the subsequent removal of the surrounding tissues by a highly aggressive corrosive solution. The corrosion casting method has been known since the beginning of the 16th century, when Leonardo da Vinci injected dissolved wax into bovine cerebral chambers (Paluzzi et al., 2007). Over the following centuries, all of the steps in the method have been improved. However, the aim has remained the same – to obtain the most accurate replica of the biological structure.

Correlating Micro-CT Imaging with Quantitative Histology 181

benzoylperoxide) polymerizes within a very short time (approximately 5 min), which makes it difficult to fill the complicated liver vasculature (Eberlová, 2012, unpublished results). After 5 minutes of stirring the resin with the catalyst, it was possible to fill a volume of approximately 20 ml before the Mercox started to harden. To prevent any vascular lesions and artifacts, the use of an infusion pump appears to be warranted (Minnich et al., 2002).

Unfortunately, Mercox does not offer sufficient X-ray-opacity for micro-CT. To fill and opacify microvessels *ex vivo*, the silicone rubber Microfil MV (Flow Tech, Inc., Carver, Massachusetts) appears to be a substance of choice (Gössl et al., 2003). The Microfil MV kit is available in five radio-opaque colors and comprises MV-Diluent, MV compound and MV Curing Agent. The working time of Microfil is approximately 20 minutes. However, Microfil MV is not suitable for conventional corrosion casting techniques using potassium hydroxide. Two alternative techniques for the subsequent tissue clearing are offered instead: alcohol-methyl salicylate clearing, which produces a stiffer tissue useful for gross

observation, and glycerin clearing, which yields a more flexible tissue and vessels.

**statistical features** 

see Fig. 4.

**6. Automatic image processing, topology analysis and measurement of** 

This chapter describes an approach to automatic micro-CT image processing using computer vision techniques and the Mercox vascular corrosion casts of the intestinal mucosa described in the previous chapter. The input data were acquired with Xradia XCT 400 (Xradia, Pleasanton, CA, USA) by scanning Mercox vascular corrosion casts. The resulting data set consisted of a DICOM stack of approximately 1000 slices, each 1000 times 1000 pixels, such that the scanned volume contained 109 volume elements (voxels), each represented by a 16-bit signed number. The depicted volume in reality was a cube with an edge of approximately 35 mm, whereas the voxel edge was 36.53 µm. For image processing, we used the Insight Toolkit library for C++ (www.itk.org) and MATLAB with its Image Processing Toolbox (The MathWorks, Inc., Natick, MA, USA). The first step of the image processing was an analysis of the density distribution of the data, using a density histogram;

Segmentation is a process in computer vision that divides the voxels of the source image into two subsets – foreground and background – depending on their affinity to the objects of the real world (Sonka et al., 1998). In our case, the foreground was formed by the vascular

Thresholding is a common and widely used image segmentation method in machine vision. This technique outputs a binary image *g(i,j,k)* that classifies the voxels of the source image

*f(i,j,k),* in which *i, j, k* represent the spatial indices of the data, according to equation 2:

1 0

*g(i, j,k ) for f (i, j,k ) T*

*for f (i, j,k ) T* 

. (2)

corrosion cast, whereas all of the other data were considered to be background.

### **5.2. Casting media**

Because the highest authenticity is required in the proportions of 3-D corrosion casts, casting media must have sufficient viscosity (to pass through but not to penetrate); they must be capable of even, rapid polymerization with minimal shrinkage and of physicochemical resistance to the subsequent corrosion and dissection procedures. All of these properties are combined in methylmethacrylate (MMA) resin. The MMA polymerizing fluid can be produced by warming the MMA monomer with a catalyst, benzoyl peroxide. MMA easily penetrates the capillary network and flows out via the opposite vessels (Kachlík & Hoch, 2008). A polyurethane pigment paste of various colors can be added into the mass along with the X-ray contrast medium, e.g., barium sulfate (BaSO4) or mercury sulfide (HgS) (Gadžijev & Ravnik, 1996). There are also commercially produced, partially polymerized MMAs (e.g., Mercox or Dentacryl), but these products vary considerably in their rheological properties. Dentacryl was used mainly in the last century, originally in dental prosthetics. This compound is a commercially produced, colorless, partially polymerized MMA that is not, due to its high viscosity, suitable for casts of the microvasculature (Kachlík & Hoch, 2008). The greatest advantage of this casting medium is its low cost; thus, Dentacryl is used for more spacious casts, such as those of the bronchial tree (Havránková et al., 1989) or paranasal sinuses (Hajnis, 1988). Painting clays can be used to color Dentacryl. Mercox is a low-viscosity acrylic resin, a MMA monomer stabilized solution. Mercox II (Ladd Research, Williston, Vermont, USA) is commercially offered in two colors (blue and red); the kit contains an MMA resin and a catalyst (benzoyl peroxide). The Mercox II system combines excellent permeability through the entire vascular bed, excellent infiltration properties, short time of preparation and polymerization, minimal shrinkage and high chemical resistance. Although its price is rather high, it is a method of choice for the vascular corrosion casts (Bartel & Lametschwandtner, 2000; Minnich et al., 2002; Lametschwandtner et al., 2004; Kachlik & Baca, 2006, Kachlík & Hoch, 2008).

In our experience with obtaining corrosion casts of porcine liver vascular trees, it is helpful to administer 50,000 IU of heparin in 1 liter of saline and subsequently rinse the vascular bed with 5 liters of Ringer´s solution prior to sacrificing the animal. We found that even the lowest Mercox dilution recommended by the manufacturer (20 ml resin/0.4 g of 40% benzoylperoxide) polymerizes within a very short time (approximately 5 min), which makes it difficult to fill the complicated liver vasculature (Eberlová, 2012, unpublished results). After 5 minutes of stirring the resin with the catalyst, it was possible to fill a volume of approximately 20 ml before the Mercox started to harden. To prevent any vascular lesions and artifacts, the use of an infusion pump appears to be warranted (Minnich et al., 2002).

180 Injury and Skeletal Biomechanics

the biological structure.

**5.2. Casting media** 

Kachlik & Baca, 2006, Kachlík & Hoch, 2008).

(Gadžijev & Ravnik, 1996), urinary tract (Marques-Sampaio et al., 2007) and lymphatic system (Fujisaka et al., 1996) have been visualized by means of corrosion casting. The steps for obtaining the corrosion casts are as follows: perfusion of the hollow target structures to remove the contents (in the case of vascular casts, prior heparinization is required for the maintenance of blood fluidity), injection of the casting media, its polymerization in the fully filled cavities and the subsequent removal of the surrounding tissues by a highly aggressive corrosive solution. The corrosion casting method has been known since the beginning of the 16th century, when Leonardo da Vinci injected dissolved wax into bovine cerebral chambers (Paluzzi et al., 2007). Over the following centuries, all of the steps in the method have been improved. However, the aim has remained the same – to obtain the most accurate replica of

Because the highest authenticity is required in the proportions of 3-D corrosion casts, casting media must have sufficient viscosity (to pass through but not to penetrate); they must be capable of even, rapid polymerization with minimal shrinkage and of physicochemical resistance to the subsequent corrosion and dissection procedures. All of these properties are combined in methylmethacrylate (MMA) resin. The MMA polymerizing fluid can be produced by warming the MMA monomer with a catalyst, benzoyl peroxide. MMA easily penetrates the capillary network and flows out via the opposite vessels (Kachlík & Hoch, 2008). A polyurethane pigment paste of various colors can be added into the mass along with the X-ray contrast medium, e.g., barium sulfate (BaSO4) or mercury sulfide (HgS) (Gadžijev & Ravnik, 1996). There are also commercially produced, partially polymerized MMAs (e.g., Mercox or Dentacryl), but these products vary considerably in their rheological properties. Dentacryl was used mainly in the last century, originally in dental prosthetics. This compound is a commercially produced, colorless, partially polymerized MMA that is not, due to its high viscosity, suitable for casts of the microvasculature (Kachlík & Hoch, 2008). The greatest advantage of this casting medium is its low cost; thus, Dentacryl is used for more spacious casts, such as those of the bronchial tree (Havránková et al., 1989) or paranasal sinuses (Hajnis, 1988). Painting clays can be used to color Dentacryl. Mercox is a low-viscosity acrylic resin, a MMA monomer stabilized solution. Mercox II (Ladd Research, Williston, Vermont, USA) is commercially offered in two colors (blue and red); the kit contains an MMA resin and a catalyst (benzoyl peroxide). The Mercox II system combines excellent permeability through the entire vascular bed, excellent infiltration properties, short time of preparation and polymerization, minimal shrinkage and high chemical resistance. Although its price is rather high, it is a method of choice for the vascular corrosion casts (Bartel & Lametschwandtner, 2000; Minnich et al., 2002; Lametschwandtner et al., 2004;

In our experience with obtaining corrosion casts of porcine liver vascular trees, it is helpful to administer 50,000 IU of heparin in 1 liter of saline and subsequently rinse the vascular bed with 5 liters of Ringer´s solution prior to sacrificing the animal. We found that even the lowest Mercox dilution recommended by the manufacturer (20 ml resin/0.4 g of 40% Unfortunately, Mercox does not offer sufficient X-ray-opacity for micro-CT. To fill and opacify microvessels *ex vivo*, the silicone rubber Microfil MV (Flow Tech, Inc., Carver, Massachusetts) appears to be a substance of choice (Gössl et al., 2003). The Microfil MV kit is available in five radio-opaque colors and comprises MV-Diluent, MV compound and MV Curing Agent. The working time of Microfil is approximately 20 minutes. However, Microfil MV is not suitable for conventional corrosion casting techniques using potassium hydroxide. Two alternative techniques for the subsequent tissue clearing are offered instead: alcohol-methyl salicylate clearing, which produces a stiffer tissue useful for gross observation, and glycerin clearing, which yields a more flexible tissue and vessels.
