*3.2. Decorin and Fibronectin Coating of the Sca*ff*olds Does Not Induce a Disadvantageous Immune Response*

The effect of DCN- or FN-coated TPCU scaffolds on immune cells was investigated in order to estimate their suitability as vascular graft material. The immune response of a combination coating was not required as the immune system would not react differently to the presence of both proteins in one coating. The performed immunological evaluation followed the normal sequence of immune activation [9], starting with PMNs that are followed by monocytes, which differentiate into macrophages at the site of injury, and finally T cells that become activated (Figure 4a).

**Figure 4.** Immune response profile of FN- and DCN-coated planar scaffolds: (**a**) Schematic overview of the analysis steps and used immune cell assays. Polymorph nuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from human blood. Monocytes were acquired from PBMCs by magnetic separation via CD14 beads. Monocytes were differentiated into M0 macrophages (MØ) by stimulation with 50 ng/mL of macrophage colony-stimulating factor (M-CSF) for 7 days. (**b**) Surface expression of activation markers CD11b and CD66b by PMNs after 4 h: Displayed are the mean fluorescence intensities (MFI) normalized to unstimulated PMNs after isolation as mean ± SEM

(standard error of the mean) for unstimulated (unstim) and lipopolysaccharide (LPS)-stimulated cells, as well as PMNs cultured on the uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds determined with flow cytometry. Kruskal–Wallis test, n = 6. (**c**) Surface expression of activation markers CD80 and human leukocyte antigen DR isotype (HLA-DR), and tumor necrosis factor alpha (TNFα) release by monocytes. Shown are the MFI normalized to unstimulated monocytes as mean ± SEM for LPS-stimulated cells as well as monocytes cultured on uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 6–8. The TNF release is depicted in ng/105 cells as mean <sup>±</sup> SEM for unstimulated (unstim) and LPS-stimulated cells as well as monocytes cultured on the uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 5. (**d**) Surface expression of activation markers CD80 and HLA-DR, and TNFα release by macrophage: Displayed is the MFI normalized to unstimulated M0 macrophages as mean ± SEM for macrophages differentiated to M1 and as well as cells cultured on uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 6–8. The TNFα release is shown in ng/10<sup>5</sup> cells as mean ± SEM for unstimulated M0 macrophages; macrophages differentiated to M1; and as well as cells cultured on the uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 6–9. (**e**) Representative SEM images of monocytes (left) and macrophages (right) on uncoated (w/o) and with biofunctionalized scaffolds (DCN and FN). Scale bars represent 50 μm. (**f**) Expression of activation markers CD69, CD25, and HLA-DR on CD3+ T cells in whole PBMC co-cultures: Shown are representative histograms (left) and the surface expression levels as MFI normalized to unstimulated T cells as mean ± SEM (right) for αCD3/αCD28-stimulated T cells (stim) as well as T cells cultured on uncoated (w/o), DCN-coated, and FN-coated scaffold. Kruskal–Wallis test, n = 6.

Initially, the expression of known PMN activation markers, the integrin CD11b, and the adhesion molecule CD66b was analyzed (Figure 4b). The normalized mean fluorescence intensity (MFI) for CD11b (stim 2.461 ± 0.3323, *p* = 0.0179; w/o 2.406 ± 0.3393, *p* = 0.0378; DCN 2.442 ± 0.3361, *p* = 0.0217; FN 2.549 ± 0.3644, *p* < 0.0090; all versus unstim 0 hours 1 ± 0) and CD66b (stim 2.372 ± 0.3875, *p* = 0.0453; w/o 2.448 ± 0.2728, *p* = 0.0414; DCN 2.431 ± 0.3041, *p* = 0.0453; FN: 2.893 ± 0.4239, *p* = 0.0073; all versus unstim 0 h 1 ± 0) was significantly increased on PMNs after LPS stimulation (positive control) and, after culture on the uncoated/coated scaffolds, compared to the level of PMNs directly after isolation (dotted line, set to 1). Additionally, PMNs on FN-coated TPCU scaffolds displayed a significantly higher CD66b expression compared with the unstimulated controls (FN 2.893 ± 0.4239 versus unstim 4 h 0.9438 ± 0.1723, *p* < 0.0345).

In a next step, monocyte responses were studied by flow cytometry analysis of the activation markers CD80 and HLA-DR (Figure 4c). The expression level for the co-stimulatory molecule CD80 was significantly upregulated only on LPS-stimulated monocytes compared with all other experimental groups (LPS 3.254 ± 0.5533 versus w/o 0.9592 ± 0.1342, *p* = 0.0143; versus DCN 0.8888 ± 0.1209, *p* = 0.0046; versus FN 0.8325 ± 0.08414, *p* = 0.0018). No significant differences in HLA-DR expression were detectable between the tested conditions. Additionally, no enhanced TNFα release of monocytes cultured on the uncoated/coated scaffolds was measured in contrast to a significantly elevated secretion in the LPS-stimulated controls compared to the unstimulated controls (LPS 0.08859 ± 0.03039 versus unstim 0.0005580 ± 0.0002111, *p* = 0.0228).

Then, macrophages (M0 type) generated in vitro by M-CSF were screened for signs of activation or polarization (Figure 4d). M0 (unstimulated) and M1 macrophages (IFNγ/LPS-stimulated) were used as control groups. Enhanced CD80 and HLA-DR expression and increase of TNFα secretion are hallmarks of pro-inflammatory M1 macrophages. There was no difference in the CD80 expression level between M0 macrophages (dotted line, set to 1) and all other experimental groups. The expression of HLA-DR by macrophages on uncoated scaffolds was significantly decreased compared with the M0 and M1 control settings (w/o 0.5220 ± 0.05753 versus M0 1 ± 0, *p* = 0.0106; versus M1 2.453 ± 1.040, *p* = 0.0049). Whereas M1 macrophages significantly elevated their TNFα release compared with M0 macrophages (M1 0.01229 ± 0.003333 versus M0 0.0002707 ± 0.00004142, *p* < 0.0001), no enhancement in pro-inflammatory cytokine release was measurable in all other experimental groups. Macrophages on the FN-coated scaffolds actually decreased their TNFα release compared with the M1 controls

(FN 0.0009826 ± 0.0004063 versus M1 0.01229 ± 0.003333, *p* = 0.0432). Complementary to the analysis of changes in surface marker and pro-inflammatory cytokine release by monocytes and macrophages, scanning electron microscopy was applied to assess the effects of co-culture on their morphology (Figure 4e). Scanning electron microscopy images were taken after the cells were cultured for two days on the different scaffold groups. Monocytes and macrophages on the DCN-coated scaffolds formed clusters of preferentially rounded cells. Macrophages cultured on uncoated or FN-coated scaffolds displayed more diverse shapes in contrast with cells grown on the DCN-coated TPCU scaffolds.

The potential activation of T cells was determined by flow cytometry analysis of known activation markers CD69, CD25, and HLA-DR [74] after culturing complete human PBMCs on either uncoated or coated scaffolds (Figure 4f). However, only anti-CD3/anti-CD28 stimulated T cells (stim; positive control) significantly elevated the expression level for CD69 (stim 7.956 ± 1.319 versus unstim 1 ± 0, *p* < 0.0001), CD25 (stim 265.6 ± 101.5 versus unstim 1 ± 0, *p* = 0.0008), and HLA-DR (stim 2.824 ± 0.3099 versus unstim 1 ± 0, *p* = 0.0001) compared with the level of the unstimulated controls (dotted line, set to 1). No significant increase in T cell activation marker expression was observed in any other experimental group.

#### *3.3. Simulation of Endothelial Progenitor Cell Homing Using Endothelial Colony Forming Cells*

3.3.1. ECFCs Show Altered VEGFR2 and PECAM-1 Expression Patterns on FN + DCN-Coated TPCU Scaffolds Under Static Culture Conditions

ECFCs were seeded on the biofunctionalized planar scaffolds and cultured under static conditions for 24 and 48 h. The amount of adherent ECFCs was significantly higher on samples coated with FN (24 h: 257 <sup>±</sup> 57 cells/mm2 versus control with 137 <sup>±</sup> 46 cells/mm2, *p* < 0.01; 48 h: 301 v 64 cells/mm2 versus control with 52 <sup>±</sup> 32 cells/mm2, *p* < 0.001) and FN + DCN (24 h: 243 <sup>±</sup> 63 cells/mm2 versus control with 137 <sup>±</sup> 46 cells/mm2, *<sup>p</sup>* <sup>&</sup>lt; 0.01; 48 h: 292 <sup>±</sup> 54 cells/mm<sup>2</sup> versus control with 52 <sup>±</sup> 32 cells/mm2, *p* < 0.001) when compared with the uncoated samples (controls) throughout the entire culture period (Figure 5a). No significant difference of adherent cells was observed between FN coating and FN + DCN coating (24 h: *p* = 0.656; 48 h: *p* = 756). DCN coating did not show any significant difference in cell density in comparison with the uncoated controls (24 h: 105 <sup>±</sup> 40 cells/mm<sup>2</sup> versus control with <sup>137</sup> <sup>±</sup> 46 cells/mm2, *<sup>p</sup>* <sup>=</sup> 0.340; 48 h: 30 <sup>±</sup> 11 cells/mm<sup>2</sup> versus control with 52 <sup>±</sup> 32 cells/mm2, *<sup>p</sup>* <sup>=</sup> 0.460).

SEM analyses revealed that the ECFCs on the control and DCN-coated TPCU scaffolds had attained a spherical shape after 24 h whereas those on TPCU scaffolds that were coated with FN and FN + DCN showed a stretched morphology (Figure 5b). Immunofluorescence staining of samples 24 h after seeding (Figure 5c,d) identified a significantly lower PECAM-1 expression in ECFCs on FN + DCN-coated samples in comparison with FN coating (0.64 ± 0.30 versus 0.90 ± 0.25, *p* < 0.05). After 48 h, this effect tended to reverse, although the difference was not significant (0.70 ± 0.15 versus 0.54 ± 0.23, *p* = 0.073). A similar and statistically not significant tendency was detected for the fluorescence intensity of vWF. No significant changes were observed in VE-cadherin or vinculin expression. VEGFR2 expression was significantly decreased in cells cultured on FN-coated scaffolds when compared with cells grown on FN + DCN-coated scaffolds after 24 h (0.64 ± 0.11 versus 0.29 ± 0.16, *p* < 0.01). After 48 h, this effect vanished (0.28 ± 0.17 versus 0.28 ± 0.15, *p* = 0.942).

**Figure 5.** Static experiments of human ECFCs on FN-, DCN-, or FN + DCN-coated scaffolds: (**a**) Attachment and proliferation of the human ECFCs after 24 h and 48 h. Cells on FN and FN + DCN coating show a significantly higher proliferation when compared with cells gown on DCN and controls. Two-tailed *t*-test, compared to controls, n = 5, n.s. = not significant. (**b**) SEM images and (**c**) Immunofluorescence staining of ECFCs 24 h after seeding on ECM protein-coated scaffolds: Cells on FN and FN + DCN show a spread morphology in contrast to DCN coating and controls. (**d**) Semiquantitative fluorescence intensity analysis (relative pixel intensity (arbitrary units)) of cells on FN and FN + DCN shows no significant difference for the endothelial cell type marker von Willebrand factor (vWF) as well as vinculin and vascular endothelial cadherin (VE-cadherin). Platelet endothelial cell adhesion molecule (PECAM-1) expression is significantly decreased and VEGFR2 expression is significantly increased on FN + DCN-coated scaffolds after 24 h. Two-tailed *t*-test, n = 6, n.s. = not significant.

3.3.2. FN + DCN-Coating Attracts ECFCs Under Dynamic Culture Conditions

After ECFC seeding under static conditions, the cell-seeded scaffolds were dynamically cultured on a roller mixer for 24 h (Figure 6a). This approach was performed to reflect more closely the in vivo conditions. The analysis of the adherent cells showed a significantly increased cell number on the FN + DCN-coated samples when compared with the controls and DCN-coated samples (5.7% ± 4.4% versus DCN coating with 1.0% ± 0.8%, *p* < 0.05 and versus control with 0.6% ± 0.7%, *p* < 0.05). The FN coating led to a nonsignificant decrease of adherent cells compared to FN + DCN coating (Figure 6b; 3.4% ± 1.5% versus 5.7% ± 4.4%, *p* = 0.226). Cells on all samples showed comparable PECAM-1 and vWF expression levels (Figure 6c). Distinct differences were observed in the cell morphology. F-actin staining helped visualizing the spread cells on the FN- and FN + DCN-coated scaffolds and cells with a more rounded morphology on the control samples and DCN-coated scaffolds (Figure 6c).

**Figure 6.** In vitro simulation of in vivo processes: ECFC attraction under dynamic conditions. (**a**) ECFCs were seeded into tubular constructs and cultured for 24 h on a roller mixer. (**b**) Adherent cells after 24 h on control scaffolds and on DCN-, FN-, and FN + DCN-coated scaffolds. FN + DCN coating shows a significantly higher cell number when compared with DCN coating and controls. One-way ANOVA, n = 4. (**c**) PECAM-1 (green), vWF (red), and F-actin (yellow) expression in ECFCs. Cells on FN and FN + DCN show a more spread morphology in contrast to the DCN and control samples.
