*2.5. Ultrasound*

The rapid development of implantable TE platforms has created a major necessity for non-invasive, non-ionizing, and non-destructive techniques for the in vivo tracking and imaging of implantable tissues. Ultrasound imaging technologies and their associated multi-modality approaches (e.g., ultrasound-photoacoustic imaging [93]) have been investigated due to their specific advantages for TE applications (Figure 6). Importantly, ultrasound techniques can enable in-situ quantitative measurements of various properties of engineered tissues, including extracellular matrix (ECM) formation, degradation, mechanical strength, cell infiltration, vascularization, and blood perfusion and oxygenation [93,94]. This is while most in vitro or ex-vivo scaffold characterization modalities, such as electron or optical microscopy, and X-ray tomography have limitations for in vivo tracking of scaffolds, due to their invasiveness, limited penetration depth (few hundred micrometers), or poor contrast. Thus, ultrasound could be an ideal tool for diverse preclinical and clinical applications.

Ultrasound utilizes sound waves at frequencies over 20 kHz (Figure 7). In a clinical setting, frequencies ranging from 1–15 MHz are used to generate images of features in biological tissues [95]. For instance, PEG hydrogels have been characterized using B-mode ultrasound to visualize varying amounts of ECM proteins and cell composition in real time over 18 days [96]. This technique utilizes a 12 MHz imaging frequency which limits the penetration, making it difficult for probing scaffolds in vivo. However, this method is suitable for validation experiments in preclinical applications. This validation method was used in a similar study, where collagen deposition was calculated in scaffolds with myofibroblasts to quantify protein concentration [94,97–99].

**Figure 7.** (**A**,**B**) Experimental setup of ultrasound used to image different areas of a scaffold made of PEG hydrogel. (**C**) Example of output image from the system. Reproduced with permission from Ref. [96]**.**

Along with quantifying biological components concentration, mechanical properties of TE constructs can be evaluated using ultrasonic modalities coupled with computational methods. Previous studies have shown that utilizing ultrasound elastography can yield measurements of elasticity and stiffness of soft tissues to determine pathological conditions such as inflammation and tumors. For instance, Walker et al., reported a strong correlation between the compressive moduli calculated from ultrasound and mechanical testing of TE cartilage [100]. Ultrasonic modalities offer robust information due to their unique interactions with biological tissues. As effective signal processing and computational methods improve, more methods are increasingly being applied to generate information-rich data sets of TE tissues from ultrasound acquisitions.

While ultrasound (sonography) methods may offer much lower resolution than MRI and CT for imaging of bioprinted constructs, they can monitor the condition of bioprinted tissues in vivo in real time readily, at much lower costs [101]. Ultrasound has recently found other novel applications in 3D bioprinting technologies, helping to tackle some of the main challenges related to this additive biomanufacturing techniques. Acoustic radiation in an ultrasound standing wave field (USWF) forces the cells and accumulates them at the pressure nodes, at low pressure areas [101]. This technique (so called ultrasound-assisted biofabrication) results in the formation of cell spheroids within minutes at relatively narrow/homogeneous size distributions. For instance, USWF was utilized to generate endothelial cell spheroids which showed enhanced neovessel formation [102]. Further, low-intensity ultrasound is reported to enhance stem cells proliferation and differentiation [103].
