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Electrospun 3D multi-scale fibrous scaffold for enhanced human dermal fibroblasts infiltration

2.9 Cell Infiltration Characterization hydrophobic nature of 2D electrospun PCL fiber mat
limits diffusion of polar fluid such as cell culture me-
Cryosectioning technique was employed to obtain the dium and cell containing solution into electrospun
information of cell infiltration. HDFs were cultured on PCL fiber mat, thereby limiting the functions of a
electrospun scaffold for 24 hours. After that, the cells
were washed with phosphate buffer saline, and then scaffold in promoting cellular infiltration and mass
fixed with 4% paraformaldehyde for 20 minutes, fol- transfer. In order to address this concern, surface
lowed by 3 times rinsing in PBS for 10 minutes. The modification was carried out in this study. Here, polar
fixed samples were embedded in tissue embedding amino group was introduced onto the fiber surface by
medium (Jung tissue freezing medium), leaving in a aminolysis. Subsequently, glutaraldehyde was intro-
fridge overnight at 4°C to allow full penetration. The duced as bifunctional linker to link proteins to fiber
samples were then frozen in liquid nitrogen and cut surface. Therefore, this allows the modified PCL na-
into 5 µm thick sections in the center part via a cryos- nofiber to couple with various hydrophilic biomole-
tat (CM3050S, Leica Microsystems, Bannockburn, cules (e.g., collagen, gelatin, peptides) that would be
IL). All the samples were placed onto glass slides recognized by cell receptor. Among all, gelatin is
coated with 1% gelatin. The nucleus of cells were chosen in this study because it is recognized as one of
stained with 4',6-diamidino-2-phenylindole (DAPI) the most cost-effective peptides with great potential to
that emitted blue fluorescence when viewed under a promote epithelization and granulation tissue forma-
fluorescent microscope (Eclipse 80i microscope, Ni- tion during wound healing [30] . After grafting with ge-
kon). Triplicates were viewed and captured for each latin molecules, the contact angle measured on 2D
scaffold type. electrospun PCL fiber mat was significantly reduced
from 116° to 46°, showing enhanced surface wettabil-
2.10 Detection of ECM Proteins Deposited by HDFs ity. This enhanced surface wettability and bioactivity

Surface modified 3D multi-scale scaffold were seeded provided by the gelatin molecules would be essential
with HDFs for 21 and 28 days. The scaffolds were to promote cell infiltration and proliferation, as well as
then immersed in Jung tissue freezing medium and nutrient exchange within the scaffold.
frozen in liquid nitrogen before kept in a –80°C freez- 3.2 Morphology and Mechanical Properties of
er. Staining of proliferation marker, Ki67 and ECM Surface Modified 2D Electrospun PCL Scaffold
proteins, including Collagen I, Collagen III, Fibronec-
tin and Elastin, were carried out according to standard The effect of surface modification on the electrospun
protocols. Positive control (mouse multi-tissue) and fibrous scaffold physical properties was examined
negative control (samples stained in the absence of with SEM and tensile testing. As shown in Figure 1,
primary antibody) were stained for comparison during SEM micrographs revealed that surface modification
immunohistochemistry study. process did not alter the electrospun fiber network
structure. Gelatin grafted electrospun fibers remained
2.11 Statistical Analysis intact with fiber diameter and arrangement similar to
Experimental data were expressed as means ± stan- pristine electrospun PCL scaffold. Tensile test also
dard deviation (SD). Student’s t-test assuming unequal demonstrated similar Young’s modulus, yield stress,
variance was used to calculate p-values, where p<0.05 ultimate tensile stress, yield strain and elongation
were considered significant. at break for both 2D electrospun PCL scaffold with
and without surface modification, as summarized in
3. Results and Discussion Table 1. It can be concluded that surface modification
process neither disrupted the fiber morphology nor
3.1 Surface Modification of 2D Electrospun PCL altered the mechanical properties of electrospun PCL,
Scaffold despite the concern of strong basicity of diamine on
When hydrophobic PCL was electrospun into a 2D the bulk mechanical property of electrospun PCL [27] .
fiber mat with high surface roughness and pores, the Even though 2D instead of 3D scaffold was used to
wettability was significantly decreased further. The demonstrate the effect of surface modifcation on the
contact angle of this 2D electrospun PCL fiber mat scaffold property, it is expected that 3D scaffold
measured with sessile drops method was 116°, show- would not behave differently because the response
ing the high hydrophobicity of the surface. The super- would be an inherent property of the material.
84 International Journal of Bioprinting (2016)–Volume 2, Issue 1