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International Journal of Bioprinting Endothelial monolayer formation on scaffolds

Figure 3. Impact of pore size on the ability of endothelial cells to overgrow pores. Confluent cell layer and closed pores were observed on 40 × 40 (A),
60 × 60 μm (B), and 80 × 80 μm pores (C). Gaps in the cell layer were observed on larger pores of 100 × 100 µm (D) and 120 × 120 µm pores (E). Typical
cobblestone-like cells in non-square pores (here 80 × 40 µm) (F). The cell nuclei are labeled in blue, the actin filaments are in red, whereas VE-cadherin
is in green. Percentage of overgrown pores as a result of the pore diameter of MEW scaffolds after 7 days of HUVECs culture (G). Data derived from
eight independent experiments (n = 8) with 100 pores in each. Statistical significance was examined using two-way ANOVA, and statistical significance is
indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001). The standard deviation is indicated by the error bars.

3.4. Characterization of the fibrin layer fiber diameter and pore size could be lowered greatly when
The fibrinogen coating modifies the MEW scaffolds’ actual compared to MEW, controlled pore sizes and shapes, as well
pore size. Antibody staining of the fibrin was used to assess as controlled fiber deposition, were not possible (Figure
the impact of the presented coating method (Figure 4A). 5A). By adjusting printing conditions, MES scaffolds with a
This exposed an irregular fibrin layer on the scaffold that pore size range of 4.88 to 55.41 µm (median 16.94 µm) were
either filled the pores of the MEW scaffold or reduced their created (Figure 5D). After fibrin coating and cultivation
size. After coating with fibrin, a percentage of 19.45% of the with endothelial cells for 7 days, chaotic cell layers with
pores was filled, and the diameter of the remaining pores many gaps were observed (Figure 5B and C).
was decreased (Figure 4B). For scaffolds with a 40 µm
interfiber spacing, the mean pore size of these remaining 3.6. Static and dynamic cell culture of endothelial
pores was 22.11 ± 2.42 µm (n = 3) after fibrin coating. cells on MEW Scaffolds
Fibrin-coated MEW/FDM scaffolds were seeded with
3.5. Melt electrospinning (MES) scaffolds HUVECs and underwent cultivation under either static (0
To evaluate the formation of endothelial layers on a dyn cm ) or dynamic conditions on two shear stress levels
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scaffold with randomly oriented fibers, MES scaffolds with 5 or 10 dyn cm (Figure 6). Under shear stress of up
2
were produced for comparison. In comparison to MEW to 10 dyn cm (≙1 Pa), a confluent endothelial monolayer
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scaffolds, fibers in MES scaffolds were randomly deposited developed on all scaffolds. After 3 days of dynamic
and formed a chaotic mesh. While fiber diameter and pore cultivation, cells on the scaffold started to show typical
size were significantly lower than those of MEW scaffolds, morphologic changes (see Figure 6A–C). Cells cultivated
controlled pore sizes and shapes, as well as controlled fiber under at least 5 dyn cm showed cell alignment with the
2
deposition, were not attainable in MES scaffolds. While flow and less rounded cell morphology in comparison to

Volume 10 Issue 1 (2024) 483 https://doi.org/10.36922/ijb.1111