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. 2020 Mar 23;9(3):778.
doi: 10.3390/cells9030778.

Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes Endothelialization in Dynamic In Vitro Culture

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Free PMC article

Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes Endothelialization in Dynamic In Vitro Culture

Ruben Daum et al. Cells. .
Free PMC article

Abstract

Appropriate mechanical properties and fast endothelialization of synthetic grafts are key to ensure long-term functionality of implants. We used a newly developed biostable polyurethane elastomer (TPCU) to engineer electrospun vascular scaffolds with promising mechanical properties (E-modulus: 4.8 ± 0.6 MPa, burst pressure: 3326 ± 78 mmHg), which were biofunctionalized with fibronectin (FN) and decorin (DCN). Neither uncoated nor biofunctionalized TPCU scaffolds induced major adverse immune responses except for minor signs of polymorph nuclear cell activation. The in vivo endothelial progenitor cell homing potential of the biofunctionalized scaffolds was simulated in vitro by attracting endothelial colony-forming cells (ECFCs). Although DCN coating did attract ECFCs in combination with FN (FN + DCN), DCN-coated TPCU scaffolds showed a cell-repellent effect in the absence of FN. In a tissue-engineering approach, the electrospun and biofunctionalized tubular grafts were cultured with primary-isolated vascular endothelial cells in a custom-made bioreactor under dynamic conditions with the aim to engineer an advanced therapy medicinal product. Both FN and FN + DCN functionalization supported the formation of a confluent and functional endothelial layer.

Keywords: bioreactor; biostable polyurethane; decorin; electrospinning; endothelial progenitor cells; endothelialization; fibronectin; tissue engineering; vascular graft.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
A newly developed polyurethane is used to produce planar and tubular electrospun scaffolds (a), which are biofunctionalized with either fibronectin (FN) or decorin (DCN) or with both extracellular matrix (ECM) proteins in combination (b,c). Besides investigating the immunology (d) and endothelial colony forming cell (ECFC) behavior on either planar (e) or in tubular scaffolds (f), the tubular scaffolds were also cultured with primary-isolated vascular endothelial cells (vECs) in an tissue-engineered vascular graft (TEVG) approach (g) in order to assess an ECM protein-improved endothelialization.
Figure 2
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.
Figure 3
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.
Figure 4
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 ± 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/105 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.
Figure 5
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.
Figure 6
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.
Figure 7
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.
Figure 8
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.

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