*2.3. Angiography*

An angiogram or arteriogram is a diagnostic procedure that uses specific dyes to outline the arteries in a patient (Figure 5). Arteries are invisible to the clinical imaging tools under normal conditions, and thus, their visualization requires utilizing some type of contrast agents. There are three main forms of angiograms, each relating to the imaging platform that is used to generate the clinical images.

Digital subtraction angiography (DSA) is the more common method of getting arterial images in the clinic. The artery to be imaged is numbed with a local anesthetic and then a contrast agen<sup>t</sup> is injected, which outlines the vascular network downstream. Following contrast introduction, X-ray is used to acquire the vasculature images [56–59]. DSA normally takes around 20 min to perform. Computer tomography (CT) angiography is another method to acquire high-resolution 3D images of a patient's vasculature. Similar to DSA, CT angiography also requires a contrast agen<sup>t</sup> introduction, but unlike DSA, the injection site is the vein in the arm, usually a drip, which allows for the entire arterial network to be imaged, if required. Image acquisition is very fast as it only takes a few seconds to generate them. The third common angiography method uses MR to generate the needed 3D vasculature images [58,60–64]. Gadolinium is the most common contrast agen<sup>t</sup> that is used with MR angiography (Figure 3). Post introduction, any artery in the body can be imaged. As with CT angiography, MR approach is a fast procedure, usually performed on the same day.

**Figure 5.** Example angiography (MR) outlining via contrasting the heart chambers and attendant vasculature. Reproduced with permission from Ref. [64].

Since angiography procedures involve the injection of a contrast agen<sup>t</sup> to generate images, there may be some risks, such as allergies to the contrast, bleeding at the puncture site, or false aneurism [65]. A rarer but serious complication can happen if there is already some kidney damage present, where contrast injection can further deteriorate kidney function. Each of these complications can be successfully mitigated via appropriate pre-procedure preparations, or with simple surgical post-procedure manipulation in the case of the false aneurysm.

Angiography is almost exclusively a clinical imaging technique, so its application to in vitro TE has been limited so far, done predominantly in excised tissue slices from mice and pig. Nonetheless, as the additive manufacturing, specifically bioprinting technologies, enter the tissue bioengineering field, complex in vitro tissue models that incorporate vascularization will require advanced visualization and tracking methods for both modeling applications in the lab and for translational applications, such as cardiac patch implants or vessel grafts post-stenosis. Having a complete picture of all sources of flow into and out of bioengineered tissue mimics would be critical to recapitulating their functionality.

### *2.4. Computed Tomography (CT)*

CT has been widely used as a biomedical imaging technique over the last decades due to its high spatial and temporal resolution. CT imaging generates a 3D reconstruction of the targeted sample by collecting the transmitted X-ray at di fferent angles using a multi-array detector (Figure 6) [12]. Since the CT contrast is sensitive to the materials that attenuate the X-ray transmission, this technique has been widely used to image tissue structures which have high mineral concentrations, like bone and the surrounding tissue. Consequently, CT has been extensively used in di fferent bone TE applications [66–69]. The development of more sensitive techniques like micro-CT has allowed for the study of the morphology and 3D structure of di fferent sca ffold geometries in the sub-micron scale as well as the tracking of di fferent cells incorporated into the sca ffold structures [70–76]. These unique advantages have also allowed CT to be used in conjunction with new additive manufacturing techniques, such as 3D (bio)printing, for di fferent implant-manufacturing purposes [66].

**Figure 6.** Cellular tracking using gold nanoparticles as a contrast agen<sup>t</sup> and imaged with CT. Reproduced with permission from Ref. [77].

A drawback of the CT technique is that it is less sensitive to visualize the contrast between di fferent soft tissue structures. However, the sensitivity can be improved by utilizing di fferent contrasting agents. Currently, di fferent biomaterials including gold [78–83], heavy elements [84], cationic agents [85], polymers [74], and nanoparticles [84,86] are being used as contrast agents in di fferent CT imaging applications ranging from animal models to clinical studies. For instance, di fferent animal model studies have employed CT with radio-transparent contrast agents like polymer [87] and alkaline-based agents [88] as in vivo imaging techniques to quantify di fferent soft tissue structures like hepatic vascular and parenchymal regeneration as well as vascular network at a capillary level. CT with contrast angiography has also been used to study the stability of human cell-derived engineered heart valve after implantation in sheep [89,90]. Clinically, CT with an iodine-based agen<sup>t</sup> has been used to study myocardial fibrosis in patients with hypertrophic cardiomyopathy [90]. These materials can be implanted in the soft tissue sca ffold, thus allowing for new in vivo tracking applications. Furthermore, these nanobiomaterials can also be used as therapeutics by including functionalized medicine through surface modification [77,91]. In addition to the development of new biocompatible contrasting materials, more sensitive detectors such as photon-counting detector technology have also been developed to enhance the visualization of di fferent soft tissues within the CT techniques [92]. As a result, contrast agents can be used both as diagnosis and therapeutics. Thus, CT imaging technologies

can be used to monitor the efficacy of drug implants in vivo due to its high spatial resolution and penetration in comparison with other imaging techniques.

More recently, CT imaging has been utilized as a nondestructive tool for longitudinal and volumetric measurement of scaffold degradation both in vitro and in vivo [81]. For this purpose, gold nanoparticles, used as contract agents, were covalently conjugated to collagen polymer during scaffold fabrication, resulting in the generation of CT-visible collagen constructs. The X-ray attenuation of the conjugated scaffolds was used to measure hydrogel degradation over the time in culture.
