*3.4. In Vitro Tissue Engineering Approach Using Vascular Endothelial Cells*

3.4.1. vECs Form an Endothelial Layer on FN- and FN + DCN-Coated Scaffolds Under Static Culture Conditions

vECs were seeded on the biofunctionalized planar constructs and cultured for 1, 4, and 7 days in order to investigate endothelialization (Figure 7a). One day after seeding, the cell number for all conditions was not significantly different. On day 4, vECs significantly increased proliferation on FN coating (78 <sup>±</sup> 26 cells/mm2 versus control with 8 <sup>±</sup> 7 cells/mm2, *p* < 0.01) and FN + DCN coating (55 <sup>±</sup> 27 cells/mm<sup>2</sup> versus control with 8 <sup>±</sup> 7 cells/mm2, *<sup>p</sup>* <sup>&</sup>lt; 0.05), while the VEC count on the DCN-coated samples had slightly decreased (7 <sup>±</sup> 5 cells/mm<sup>2</sup> versus control with 8 <sup>±</sup> 7 cells/mm2, *p* < 0.931). This trend continued until day 7, on which a significantly increased cell count was detected for FN coating (186 <sup>±</sup> 47 cells/mm2 versus control with 16 <sup>±</sup> 16 cells/mm2, *p* < 0.001) and FN + DCN coating (135 <sup>±</sup> 50 cells/mm2 versus control with 16 <sup>±</sup> 16 cells/mm2, *p* < 0.01) in comparison with the uncoated controls. DCN coating of the TPCU scaffolds showed no improvement when compared with the control samples. Over the entire period of the experiment, the cell count was not significantly different between FN and FN + DCN coating.

**Figure 7.** Static cell culture experiments of vECs on FN- and DCN-coated scaffolds: (**a**) Attachment and proliferation of vECs after 1, 4, and 7 days. vECs on FN and FN + DCN coating show a significantly higher proliferation rate compared with cells gown on DCN coating or control scaffolds. Two-tailed *t*-test, compared with control samples, n = 3, n.s. = not significant. (**b**) SEM images and (**c**) IF staining of vECs 7 days after seeding on ECM-coated scaffolds. Cells on FN and FN + DCN coating show a spread morphology in contrast with cells on DCN coating and control samples. (**d**) Semiquantitative fluorescence intensity analysis (relative pixel intensity (a.u.)) of cells on FN and FN + DCN coating shows no significant difference for PECAM-1, vWF, vinculin, or VE-cadherin expression. Two-tailed *t*-test, n = 5, n.s. = not significant.

While vECs on the control and DCN-coated scaffolds showed a spherical shape after 7 days as assessed using SEM, on FN and FN + DCN-coated scaffolds, vECs were stretched out and formed an almost confluent endothelial cell layer (Figure 7b). IF staining confirmed the expression of the endothelial cell type-specific markers PECAM-1, vWF, and VE-cadherin in the vECs on both FN and FN + DCN coating (Figure 7c). Semiquantitative analysis of fluorescence intensities revealed no significant differences of marker expression between FN and FN + DCN coating (Figure 7d). Vinculin expression was comparable in vECs on both coatings. With regard to VEGFR2, an increased fluorescence intensity in cells grown on the FN + DCN-coated samples was observed. However, due to a high variation in expression levels of individual experiments, no statistical significance between cells grown on FN or FN + DCN coating could be determined.

In summary, our data showed that DCN coating of the TPCU scaffolds did not have a substantial advantage when aiming for an increased VEC proliferation or an improved cell–cell or cell–material interaction. For this reason, only FN biofunctionalized TPCU scaffolds were used for the following in vitro tissue engineering experiments.

3.4.2. vECs Cultured in a Custom-Made Bioreactor Under Flow Form a Confluent and Aligned Cell Layer on FN-Biofunctionalized TPCU

After successful implementation of the developed bioreactor system, we aimed to test whether the FN-biofunctionalized TPCU scaffolds can be endothelialized under dynamic conditions. vECs were seeded into the tubular TPCU scaffolds, and after an initial culture for three days under static conditions to allow cell attachment, a flow was employed that was stepwise increased to 25 mL/min within 1.5 days (Figure 2e). Under this flow, which causes a shear stress of about 0.03 Pa, the vEC-seeded FN-biofunctionalized scaffolds were cultured for seven days. Metabolic activity assessment using an MTT assay showed that a large part of the inner wall of our construct was covered with living cells, as indicated by the purple formazan stain (Figure 8a). IF staining and SEM further revealed a layer of confluent vECs that were aligned in the direction of flow (Figure 8b,c).

**Figure 8.** Tissue-engineering approach with vascular endothelial cells cultured for 7 days on FN-biofunctionalized electrospun tubular TPCU scaffolds under dynamic conditions: (**a**) Inner wall of the tubular construct shows living vECs indicated by the purple formazan stain. (**b**) PECAM-1, vWF, VE-cadherin, vinculin, VEGFR2, and F-actin expression were detected. vECs show an aligned morphology. (**c**) SEM confirms vECs that had aligned with the flow to which they were exposed to during the dynamic culture in the bioreactor.

We confirmed the expression of the endothelial cell markers PECAM-1, vWF, and VE-cadherin. However, PECAM-1 and VE-cadherin did not appear to be located on the cell membrane as usual. Vinculin and VEGFR2 were also detected in the cells. Nevertheless, the staining of VEGFR2 showed only a weak signal.

#### **4. Discussion**

Due to a proven biocompatibility and biostability at body temperature [55,57], we selected for this study a novel thermoplastic polycarbonate urethane for the fabrication of a TEVG. At first, scaffolds were produced by electrospinning of the TPCU and were disinfected with 70% ethanol. Microbiological studies showed that ethanol treatment did not achieved 100% sterility (Figure S3; 2 out of 9 plates showed germ growth). We are aware that disinfection with ethanol does not necessarily inactivate all forms of microorganisms [75]; therefore, for the clinical translation, a more efficient sterilization method should be considered.

After disinfection, scaffolds were then biofunctionalized by adsorption of FN and DCN, either alone or in combination. The adsorbed proteins did not impact elastic modulus or burst pressure of the tubular constructs (Figure 3). We demonstrated that the biomechanical properties of our constructs were comparable to native vascular tissue (Table 2).

The ability to mimic the nanofibrous topography of the ECM makes electrospinning a powerful method for cardiovascular tissue-engineering applications. Several studies have already described the influence of fiber and pore size on cell adhesion, cell migration, proliferation, and differentiation, as well as cell–cell interaction [76–78]. In native blood vessels, the ECs are located on the basal lamina, a mixture of defined ECM proteins that form a network and bind cells [79]. The literature describes a wide range of pore and fiber diameters (1–1000 nm) from different vessels, depending on the position and physical properties of the vessel [80]. The main collagen component of the basal lamina is collagen type IV. It forms fibers that range from 20 to 52 nm [80–82]. In our study, the fiber diameters were between 699 ± 61 nm and 776 ± 163 nm, which is much higher compared to the collagen type IV fibers in native vessels. However, other studies developing electrospun vascular grafts reported comparable [83] or even larger fiber sizes [84,85] on which a functional endothelium was formed [84]. The pore size strongly depends on the vessel type and ranges between 5 nm and 8 μm [80,82,86–89]. Our constructs showed pore sizes between 0.08 <sup>±</sup> 0.01 <sup>μ</sup>m2 and 0.12 <sup>±</sup> 0.05 <sup>μ</sup>m2, which lies in the range of a native vessel.

Several studies have already described that FN improves the endothelialization of vascular grafts [19,48,49,51]. In our study, we observed a fibrous-like structure of the coated FN (Figure 3b). This phenomenon can be interpreted as material-driven fibrillogenesis, first described by Salmeron-Sanchez et al. [3]. In the human body, FN matrix assembly is a cell-mediated process [90] that influences cell growth, cell differentiation, and cell–cell interaction [76–78,90,91]. It has been shown that the adhesion of FN on poly (ethyl acrylate) (PEA) can lead to a spontaneous organization of FN into protein networks. It has also been shown that cell-free material-induced FN fibrillogenesis influences the maintenance and differentiation of stem cells [3,92]. Furthermore, it was described that the FN network has an increased ability to store growth factors [93]. To the best of our knowledge, our study is the first to show that material-driven fibrillogenesis can be observed on electrospun TPCU fibers. We presume that the surface properties, such as hydrophobicity and polarity, are comparable to those of PEA. Whether the FN network has a significant advantage in terms of cell behavior or growth factor binding compared to dispersed, coated FN molecules would need further investigation.

In addition to FN coating, in this study, we also used DCN coating. We observed that, after coating on the TPCU, DCN was randomly distributed in aggregates on the fibers (Figure 3b). Since DCN does not form fibrils, this coating behavior was expected. Even larger, globular DCN aggregates were observed on the FN + DCN samples (Figure 3b,d). Interestingly, these aggregates were predominantly seen on the FN fibrils and not on the TPCU itself. It is known that DCN interacts with FN [94,95]. Furthermore, the interaction of proteins with materials is determined by the geometrical, chemical,

and electrical properties of the substrate [96]. In this respect, it can be hypothesized that the DCN prefers the FN surface more than the hydrophobic polyurethane surface. Interestingly, we observed a significantly increased swelling ratio for FN + DCN (Figure 3e). This was not the case with individually FN- or DCN-coated TPCU. Depending on the surface properties of the material and the interaction with other proteins, the conformation, orientation, and bioactivity of a protein can also be influenced [96–98]. With this in mind, one can assume that both DCN and FN in combination can have a different bioactivity [99].

In contrast to our previous findings using poly (ethylene glycol) dimethacrylate-poly (L-lactide) (PEGdma-PLA) or a blend of poly-ε-caprolacton and gelatin [25,100], we identified a cell-repellent effect of the DCN-coated TCPU electrospun scaffolds for both human ECFCs and human vECs. As already discussed, cells prefer to adhere to hydrophilic surfaces [101]. Since the TCPU itself is highly hydrophobic (control: 98.4 ± 3.7 ◦), it can cause a cell-repellent effect. DCN alone was not able to diminish this effect (Figure 5a,b). Cell adhesion is influenced by cell-adhesive peptides such as the RGD sequence. Since DCN does not contain these sequences, as it is the case with FN, we assume that at least this integrin-based cell–material interaction cannot be mediated by DCN. It has been described that DCN can even partially inhibit cell adhesion; however, this has only been observed with fibroblasts and not with endothelial cells [28,102]. Hinderer et al. observed an attraction of ECFCs to DCN-coated PEGdma-PLA [25]. A direct comparison with this study is therefore difficult, since this polymer has different surface properties, which influence the amount and orientation of the adsorbed DCN and thus may have an altered impact on cell behavior [96]. FN coating reversed the cell-repellent effect of the TCPU, both with and without DCN (Figure 5). We can therefore conclude that the cell attraction and proliferation is supported by FN but not affected by DCN [99,103].

Scaffolds should in general exhibit a low immunogenicity and at the same time support tissue regenerative processes. The evaluation of the immune response profiles of the analyzed control and ECM-coated scaffolds excluded any major adverse effects, with only minor innate activation characteristics. Co-culturing PMNs, as the first cells of an innate immune response, induced an activated cell phenotype regarding the expression of CD11b and CD66b. Monocytes were incompletely activated after co-culturing with the scaffold as indicated by only a weak tendency to upregulate the HLA-DR expression and to increase their TNFα release. From the literature, it is well known that the upregulation of CD80 and HLA-DR would be a hallmark of M1 macrophages [62,104] and that the fiber and pore size of electrospun scaffolds could impact the macrophage polarization state [105]. When analyzing the potential impact of the TPCU scaffolds on macrophage polarization, no clear trend to drive the process into a specific macrophage subtype could be determined. Also, the coating by either DCN or FN did not trigger a specific type of macrophage polarization. In contrast, co-culture studies with soluble recombinant DCN demonstrated that macrophages responded with an upregulated CD80 expression as well an increased secretion of TNFα and IL-10 [25]. The absent responses in the present study may result from the far lower amount of protein present on the coated scaffolds in comparison with the high protein amounts available within solutions or even by conformational changes. Not surprisingly, adaptive T cell responses were also not detected. T cells on scaffolds simply showed a trend to upregulate CD69 and HLA-DR without significant changes.

A functional endothelium is mainly characterized by cell–cell junctions [106]. As PECAM-1 is the most abundant component of the EC junction, which contributes to the maintenance of the EC permeability barrier, its expression is essential for a functional EC layer [107]. In our study, the ECFCs on FN coating revealed a significantly increased PECAM-1 expression after 24 h compared with ECFCs cultured on FN + DCN-coated scaffolds. In contrast, the VEGFR2 expression was significantly decreased in the ECFCs on FN coating after 24 h compared with FN + DCN coating. It has been reported that VEGFR2 is highly expressed in early endothelial precursor cells but not in all mature ECs [108,109]. For example, PECAM-1 is less expressed in endothelial progenitor cells, as it is typically associated with a more mature EC phenotype [110]. Interestingly, DCN has been reported to stimulate the maintenance of undifferentiated progenitor cells [111], and FN promotes endothelial

cell differentiation [112]. Therefore, we hypothesize that the FN + DCN coating in our experiments kept the ECFCs in a precursor cell state compared with the culture on only FN. It may also be possible that a direct interaction of DCN with VEGFR2 leads to its upregulation. A positive feedback loop between VEGF and VEGFR2 has been described [113]. Whether DCN has the same effect remains to be confirmed.

Since DCN exerts many other functions, an indirect regulation of VEGFR2 is also conceivable [34,114]. Mazor et al. showed that the matrix metalloproteinase-1 (MMP-1) promotes the expression of VEGFR2 [115]. The core protein of DCN in turn is able to stimulate the expression of MMP-1 [116,117]. Furthermore, Murakami et al. reported that increased concentrations of the fibroblast growth factor (FGF) led to an increase in VEGFR2 levels [118]. DCN, in turn, can bind to FGF and can increase its activity [119]. It was also described that VEGFR2 expression is regulated by the disruption of the c-MET receptor tyrosine kinase [120]. As an antagonistic ligand of c-MET, DCN is able to inhibit its activity and thus might indirectly promote VEGFR2 expression [38]. We have already discussed the hypothesis that DCN in interaction with FN may exhibit an altered bioactivity. This would explain why DCN, which was adsorbed on the TPCU scaffold surface, impacted ECs in combination with FN but did not without [96–98]. The reason for VEGFR2 upregulation can also be due to FN. It might be possible that, in combination with DCN, its conformation and function is also changed [96–98]. It has been shown that conformational remodeling of the FN matrix selectively regulates VEGF signaling [121]. VEGF in turn regulates VEGFR2 expression [113]. By binding to VEGF, FN can promote full phosphorylation and activation of VEGFR2 [122]. Interestingly, after 48 h, the difference between FN and FN + DCN coating for both the PECAM-1 and VEGFR2 expression had vanished (Figure 5d). With regard to VEGFR2, a short half-life of the receptor is described, which enables ECs to adapt quickly to changes in the extracellular environment [118,123]. This leads to the question of how long the biofunctionalized DCN coating was fully biologically active in our study. Due to its natural presence in the body, it can be easily degraded [124]. We showed that DCN acts on ECFCs for at least 24 h under static conditions. The culture of vECs over 7 days under static conditions revealed the same expression of PECAM-1 and VEGFR2 on FN and FN + DCN coating (Figure 7). This observation supports the assumption that the DCN was only active for a short period of time and that its effect had disappeared after 7 days. In addition, it is possible that the vECs are not as sensitive to DCN, as we have observed with the ECFCs. Several studies have described an increase in VEGFR2 expression during differentiation and expansion of endothelial progenitor cells [109,125]. At the same time, VEGFR2 expression was relatively low during the proliferation phase [126]. Since the vECs are mature cells, it can be assumed that the externally changed conditions do not affect the VEFGR2 expression significantly. Nevertheless, in this study, we successfully showed that vECs formed an endothelium on biofunctionalized FN-coated constructs after 7 days of culture whereas DCN-coated TPCU scaffolds did not show a significant effect on cell proliferation.

In our TEVG experiments using a custom-made bioreactor, we observed a unidirectional cell orientation in the direction of the flow. The response of ECs to shear stress is well studied [127–129]. It has been shown that, under flow, the morphology of vECs changes from a cobblestone (static) to an elongated form and that vECs align in the direction of the flow in only 24 h [127]. The hemodynamic forces can modulate not only the phenotype but also the gene expression of the cells. In this context, the correct flow is of great importance for a properly functioning endothelium [130]. In our study, IF staining revealed the expression of vWF, PECAM-1, and VE-cadherin. However, PECAM-1 and VE-cadherin were not located on the cell membrane as usually seen. VEGFR2 expression was quite weak, and the F-actin staining revealed a rather fibroblast-like cell morphology. We hypothesize that the vECs underwent endothelial-mesenchymal transition (EndMT). ECs, which undergo EndMT, lose the expression of the characteristic surface endothelial markers PECAM-1, VE-cadherin, and VEGFR2 [39,131,132]. Mahmoud et al. showed that the EndMT can be induced under low shear stress (0.4 Pa) [133]. In our approach, the cells experienced a wall shear stress of about 0.03 Pa, which is slightly lower than a venous wall shear stress (0.06 Pa) [134]. In silico simulations of our dynamic bioreactor culture

confirmed laminar flow conditions along a large part of the vascular wall using the applied parameters. Another reason for the fibroblast-like phenotype could be that ECs are highly plastic [135,136]. Therefore, culturing ECs in vitro in an artificial environment can lead to cell dedifferentiation [136,137]. This highlights the importance of fine-tuning the culture conditions to create a functional TEGV.
