*2.9. Tissue Culture of Vascular Endothelial Cells Under Dynamic Conditions*

Tubular electrospun scaffolds were cut to 7.5 cm length and biofunctionalized with 20 μg/mL FN as described previously. After inserting the graft frame into the culture chamber, 2 <sup>×</sup> 106 vECs/mL were seeded into the tubular scaffold. In order to achieve homogeneous cell adhesion across the entire tube, the culture chamber was placed horizontally and rotated every 15 minutes over 45 ◦ for 3 h at 37 ◦C and 5% CO2. The culture chamber was consecutively connected to the rest of the bioreactor setup and filled with 70 mL culture medium, supplemented with 1% penicillin-streptomycin and 1% PrimocinTM (ant-pm-1, InvivoGen, San Diego, CA, USA). The seeded cells were allowed to proliferate under static conditions during the first three days, after which the flow rate was slowly increased over the course of two days, as shown in Figure 2e. Subsequently, the tubular construct was cultured under constant flow for seven days.

**Figure 2.** (**a**) A cross-sectional schematic representation of the culturing chamber and its parts. The wireframe model on the right is overlaid by the results of an in silico simulation and shows the flow velocity when the system is perfused with a flow rate of Q = 20 mL/min. (**b**) This photograph shows the graft frame (without scaffold), once it is taken out of the culturing chamber. (**c**) A schematic representation of the entire bioreactor setup, showing the circulation and connections to the medium reservoirs and pressure buffer/bubble trap. (**d**) A photograph showing the assembled bioreactor setup with all the components for the intraluminal circulation. (**e**) Applied perfusion flow speed as function of time with the corresponding wall shear stress.

#### *2.10. Immunofluorescence Staining*

In order to examine the protein coating, the biofunctionalized scaffolds were stained using DCN mouse monoclonal IgG1 (1:200; sc-73896, Santa Cruz Biotechnology, Dallas, TX, USA) and FN polyclonal rabbit IgG (1:500; F3648, Sigma-Aldrich) antibodies. For fluorescence labeling, AlexaFluor 488 anti-mouse IgG (1:250; A-11001, Thermo Fisher Scientific) and AlexaFluor 546 anti-rabbit IgG (1:250; A-11035, Thermo Fisher Scientific) were used as secondary antibodies.

Cells cultured on the scaffolds were stained as follows: after washing once with PBS, the cell-seeded scaffolds were fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich). In order to reduce nonspecific binding, the samples were incubated with 2% goat serum-containing block solution for 30 min. Afterwards, the cells were incubated over night at 4 ◦C with the following antibodies: Vascular endothelial cadherin (VE-cadherin) monoclonal mouse IgG2B (1:500, MAB9381, R&D systems, Minneapolis, MN, USA), VEGFR2 polyclonal rabbit IgG (1:75, ab2349, Abcam, Cambridge, UK), PECAM-1 monoclonal mouse IgG1 (1:100, sc-71872, Santa Cruz), von Willebrand factor (vWF)

polyclonal rabbit IgG (1:200, A0082, Dako, Glostrup, Denmark), and vinculin monoclonal mouse IgG1 (1:500, MAP3574, Millipore, Burlington, MA, USA). F-actin was stained for 45 min in the dark with Alexa Fluor 647 Phalloidin (1:500, A22287, Thermo Fisher Scientific). Subsequently, samples were incubated with the appropriate secondary antibodies (AlexaFluor 488 anti-mouse IgG, AlexaFluor 546 anti-rabbit IgG, and AlexaFluor 488 anti-mouse IgG2b (all 1:250; Thermo Fisher Scientific)).

Finally, nuclei were stained with DAPI (1:50) for 15 min in the dark. Images were obtained by using a fluorescence microscope (Cell Observer, Carl Zeiss AG, Oberkochen, Germany).

#### *2.11. Examination of the Cell Coverage on the Tubular Sca*ff*olds*

The cell coverage of the inner wall of the tubular constructs was investigated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (M2128-1G, Sigma-Aldrich). After culturing with vECs, the constructs were incubated for 20 min with 1 mg/mL MTT at 37 ◦C and 5% CO2. The insoluble purple formazan produced by the cellular reduction of MTT was then examined macroscopically.

#### *2.12. Image Analysis*

FN and DCN coating were quantified by measuring the relative pixel intensity (RPI) of the immunofluorescence images. To assess protein expression in the experiments, the area within a defined fluorescence intensity threshold was measured and normalized to the cell number. The cell count in the static experiments was quantified by counting the DAPI-stained cell nuclei per area. The quantification of the adherent ECFCs in the dynamic experiment was performed by measuring the DAPI-stained area normalized to the total area. All images were analyzed using ImageJ [58].

#### *2.13. Scanning Electron Microscopy of Cells*

Prior to SEM imaging of the scaffolds with cells, a critical point drying step was performed. First, cells were fixed for 60 min with 4% paraformaldehyde (PFA)/ 25% glutaraldehyde in PBS. Subsequently, a series of ethanol solutions in ascending concentration up to 100% was carried out to remove water. Critical point drying was done with a CPD 030 (Bal-Tec AG, Balzers, Liechtenstein) according to the manufacturer's protocol. Prior to imaging, the specimens were platina-coated (SCD050, Bal-Tec AG) for one minute at 0.05 mbar and rinsed with Argon after the coating process. SEM imaging was performed with a SU8030 (Hitachi, Tokyo, Japan) and an Auriga® 40 (Zeiss, Oberkochen, Germany).

For SEM imaging of the monocytes and macrophages, the cells were cultured for two days on uncoated (w/o), DCN- or FN-coated scaffolds, followed by preparation (as described in Reference [62]) and imaging with a JCM 6000 Benchtop (JEOL, Freising, Germany).

#### *2.14. Statistical Analysis*

Except stated otherwise, data are presented as mean ± standard deviation. For the immune data, GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used to determine statistical significance between two groups using a one-way ANOVA/Kruskal–Wallis test. For the other data, a one-way ANOVA/Fisher's Least Significant Difference test was performed. A Welch's t-test was performed to compare between two data groups using OriginPro (OriginLab, Northampton, MA, USA). Probability values of 95%, 99%, 99.9%, and 99.99% were used to determine significance.

#### **3. Results**

#### *3.1. Biofunctionalization Does Not Impact the Mechanical Properties of Electrospun Tubular Constructs*

Electrospinning was used to fabricate 110-mm long tubular scaffolds with an inner diameter of 5 mm and a thickness of 0.40 ± 0.06 mm (Figure 3a). In order to modulate the cell–material interaction, the surface was biofunctionalized with FN, DCN, or FN + DCN. The impact of the biofunctionalization on the morphological and mechanical properties of the material was investigated (Figure 3). Fiber and

pore size analysis of the SEM images revealed no significant alteration due to protein adsorption (Figure 3e). Higher magnifications of the SEM images showed distribution of the proteins on the fibers. While DCN formed randomly distributed aggregates on the TPCU scaffolds, FN coating showed a network-like deposition in the nanometer range, which was also seen in the FN + DCN-coated samples, in which clearly recognizable aggregates were deposited on the protein network (Figure 3b, white arrows). Biofunctionalization utilizing both proteins individually and in combination was confirmed by IF staining. DCN IF staining revealed a more heterogeneous distribution of DCN in combination with FN than alone (Figure 3c, white arrows). The contact angle of the scaffolds was not significantly changed by the adsorption of either FN or DCN in comparison with the uncoated scaffolds. A significantly higher swelling ratio was observed of scaffolds that had been coated with FN + DCN (Figure 3e; control: 93.7% ± 7.7% versus FN + DCN: 117.1% ± 8.7%, *p* < 0.05). Overall, biofunctionalization had no significant influence on the mechanical properties (Figure 3e). The ultimate tensile strength ranged from 21.1 ± 3.5 MPa (DCN) to 22.1 ± 3.7 MPa (FN). Burst pressures were in the range between 3124 ± 466 mmHg (FN + DCN) to 3326 ± 78 mmHg (controls). Interestingly, the elastic modulus of the samples coated with FN + DCN showed a lower value compared to the controls, although this was not statistically significant (3.7 ± 0.5 MPa FN + DCN versus 4.8 ± 0.6 MPa controls, *p* = 0.125).

We compared the mechanical properties (elastic modulus and burst pressure) of our electrospun scaffolds with autologous grafts, which are today's gold standard for vascular bypass surgeries, using data obtained from literature (Table 2) [65]. The elastic modulus of our constructs (4.8 ± 0.6 MPa) was slightly higher than that of saphenous veins (2.25–4.2 MPa) [66,67] and of iliofemoral arteries (1.54 MPa) and veins (3.11 MPa) [68]. However, compared with an internal mammary artery (8 MPa) and a femoral artery (FA, 10.5 MPa)—used for popliteal bypass surgery—our engineered scaffolds showed a lower elastic modulus [66,69,70]. Regarding the burst pressure, engineered scaffolds (3326 ± 78 mmHg) lied within the range of a saphenous vein (1250–3900 mmHg) [66,67,71,72] and an internal mammary artery (2000–3196 mmHg) [66,71]. Konig et al. recommends for a TEGV a minimum burst pressure of 1700 mmHg [71]. We can therefore argue that our constructs have suitable mechanical properties to serve as a vascular graft or TEGV.


**Table 2.** Mechanical properties of the electrospun constructs and native blood vessels.

**Figure 3.** Morphological and mechanical characterization of the tubular biofunctionalized scaffolds: (**a**) Electrospun tubular scaffolds were fabricated with a length of 110 mm, an inner diameter of 5 mm, and a thickness of 0.40 ± 0.06 mm. (**b**) SEM images of control and biofunctionalized scaffolds: Scaffolds coated with FN show a network-like structure on the fibers. Aggregates deposited on the FN + DCN-coated samples are indicated by white arrows. (**c**,**d**) The coating of FN, DCN, or FN + DCN in combination was confirmed with IF staining: FN (red) and DCN (green). The white arrows indicate aggregates deposited on the FN + DCN-coated samples. Two-tailed *t*-test vs. control, n = 3, RPI = relative pixel intensity. (**e**) Fiber and pore size analysis shows no significant difference between the biofunctionalized scaffolds and the controls. Mechanical properties are not influenced by the protein coating. One-way ANOVA, n = 4, *p* < 0.05 vs. control.
