**3. Results**

### *3.1. Fluid-Structure Interaction Simulation*

The mechanical properties of biomimetic constructs under the e ffect of actual human body conditions are critical in terms of the clinical applications and potential failure. In the human body, blood vessels usually undergo very large blood pressure, especially in the case of a large-diameter vessel. Biomaterials such as gelatin usually undergo very large strains under loading, and the stress-strain relationship is generally nonlinear. The behavior of the TEVG is influenced not only by its geometry and material properties, but also by the fluid passing through it. Numerical simulations were carried out to estimate the displacement of the wall of the TEVG under the influence of the blood flow.

The inclusion of surrounding cardiac tissue is also essential for the FSI simulation process, because during the expansion process of the TEVG's wall the cardiac muscle would exert a tethering e ffect on the wall and thus restrict the radial motion. Hence, the integrated simulation model is more desirable than the one consisting solely of the TEVG, thus providing more realistic boundary conditions for the simulation.

The total displacement of the TEVG and cardiac muscle at the peak pressure (t = 0.5 s) is shown in Figure 6a,b, respectively. The maximal displacement of 1.18 μm occurs at the bifurcated location. The displacement in the bifurcated area is large since this is the location where the blood flow impinges on the wall of the construct, leading to a local high-pressure region. No rupture was observed from the results, implying that the risk of failure is low under coronary blood flow conditions. The compliance of the vascular construct is calculated to be 0.26%, which is within the normal range of a human blood vessel [36].

**Figure 6.** (**a**) The total displacement of the TEVG at the peak load (t = 0.5 s); and (**b**) the total displacement of the whole construct at the peak load (t = 0.5 s).

Additionally, for the time-dependent analysis, the total displacements of the TEVG in a complete heart beating cycle (0–1 s) are shown in Figure 7a–c. It could be observed that the peak displacement of the structure is still mainly located in the bifurcated region. The simulation results can be used to examine the risk of failure for the fabricated TEVG when it is integrated in other three-dimensional tissue constructs.

**Figure 7.** The time variation of the total displacement of the TEVG at: (**a**) t = 0 s; (**b**) t = 0.5 s; and (**c**) t = 1 s.

### *3.2. The Morphology of the Proposed TEVG*

In this study, a novel method of fabricating biodegradable multilayered TEVGs was proposed. The fabricated enzymatically-crosslinked gelatin vascular construct is shown in Figure 8. The top view (Figure 8a) of the construct confirmed its bifurcated structure in the three-dimensional space. To reveal the layered structure of the construct, blue and red acrylic paints were added in the hydrogel of each layer when fabricating the construct. It can be observed that the TEVG possesses a distinct multilayered structure (Figure 8b, blue: outermost layer of the structure; and red: middle layer of the structure). The inner channel of the construct became visible after being immersed in water (Figure 8c), which demonstrated its connectivity. The section view of the TEVG revealed that the molding generated a real circular shape after sacrificing the F-127 (Figure 8d) and form a bifurcated channel ready for liquid perfusion (Video S1). The innermost layer partially deviated from the center, which was supposed to be affected by the die clearance. Compared with previously seen TEVGs, the proposed TEVGs possess a branched structure while keeping the multilayered feature. This is of particular importance because over-simplified in vitro TEVG models have a limited significance for simulating the actual in vivo environment.

**Figure 8.** Morphology of the TEVG: (**a**) top view; (**b**) multilayered structure of the TEVG (blue: outermost layer of the structure; and red: middle layer of the structure); (**c**) inner channel of the construct after being submerged in water. (Scale bars: 5 mm); and (**d**) section view of the TEVG (Scale bar: 500 μm).

### *3.3. The Results after Cells Seeding*

One of the most critical properties of biomimetic structures are their in vitro cytocompatibility, and the creation of a continuous monolayer of ECs in the inner channel is necessary for the proper function of TEVGs [37]. To investigate the in vitro cytocompatibility of the fabricated TEVGs, HUVECs were seeded and statically cultured in the inner channel. The microscopic morphology image (Figure 9a,c) and the fluorescent image (Figure 9b,d) of endothelial cells after 4 h of attachment were shown. It could be observed that the HUVECs adhere well on the lumina. However, the cell distribution at this stage is quite nonuniform, and this might be caused by the uneven distribution of the cell in the suspension at the initial stage. Additionally, because the inner diameter of the channel is quite

large, the adhesion of the cells is difficult. Many cells entering from the inlet didn't have the chance to adhere and were flushed away from the outlet. After 72 h of culturing, as could be observed from Figure 9e–h, the amount of HUVECs had significantly increased, and the cells in the inner channel were found to have well spread on the wall and to have taken a normal cellular phenotype. In spite of the nonuniformity of the distribution in the attachment phase, the cells still covered the surface of the channel and formed an endothelialized monolayer. The results indicated that the crosslinked TEVG had a good in vitro cytocompatibility and was suitable for cell attachment and for proliferation.

**Figure 9.** Microscopic images and fluorescent images of cells; the dotted lines were used to divide the inside and outside of the channel: (**<sup>a</sup>**,**b**) after 4 h of attachment at the bifurcate area of the TEVG, (**<sup>c</sup>**,**d**) after 4 h of attachment at the linear channel of the TEVG, (**<sup>e</sup>**,**f**) after 72 h of culturing at the bifurcate area of the TEVG, (**g**,**h**) after 72 h of culturing at the linear channel of the TEVG. (Scale bars: 500 μm).

### *3.4. Mechanical Properties of the Proposed TEVG*

The mechanical properties of tissue-engineered biological constructs determine their application fields. The TEVGs fabricated in this study possess a high aspect ratio, which determines their susceptibility to bucking when suffering from a compressive load. Therefore, the uniaxial compressive mechanical properties of the fabricated TEVG under room temperature and physiological conditions were investigated. The mechanical properties of gelatin samples with and without crosslinking were evaluated. Figure 10a shows the compressive stress-strain curves for up to a 60% strain. The compressive modulus of the four groups of samples are shown in Figure 10b. It can be observed that the addition of mTG (1.4%, wt) increased the compressive modulus of the constructs by more than three times, from 1.5 MPa to 5 MPa at room temperature. Under 37 ◦C, the mechanical properties of the gelatin/mTG TEVG is almost the same as that under 23 ◦C, while the pure gelatin one completely solved. The main reason for this mechanical enhancement lies in the fact that the composition of gelatin includes glutamine and lysine. The presence of these amino acids means there are more crosslinking sites in gelatin, thus promoting the chance of creating stiffer gels when provided with sufficient amounts of mTG under proper crosslinking conditions. Compared with the TEVG composed of GelMA hydrogels, the maximum compressive modulus of the gelatin/mTG based TEVG is enhanced more than ten-fold [38].

The mechanical properties of the enzymatically-crosslinked TEVG are still relatively low compared to those of native blood vessels. However, as was envisioned, the real value of the fabricated vessel-mimic structure lies in its capacity to carry various types of vascular cells and to eventually develop into function blood vessels. Hence, the capability of maintaining its original morphology when integrated with other engineered scaffolds is sufficient for the proper function of the fabricated TEVG.

**Figure 10.** Compressive testing. (**a**) stress-strain curves of the samples with or without crosslinking at 23 ◦C and 37 ◦C respectively; and (**b**) compressive modulus of the samples with or without crosslinking at 23 ◦C and 37 ◦C respectively.
