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Near-field electrospinning of a polymer/bioactive glass composite to fabricate 3D biomimetic structures

a b c d

e f g

Figure 2. Near-field electrospinning of polycaprolactone (NFES) +B3 glass composite. (a) Fiber deposition control with different NFES
fabrication parameters, (b) printing schema, (c) after printing one raster in both 0° and 90° orientation, (d) magnified image showing the
fiber dimensions, (e) 10-layer scaffold, (f) magnified image of the 10-layer scaffold, and (g) cancellous bone microstructure (reproduced
with permission ).
[16]
a b can be observed in PCL + B3 glass composite scaffolds
(Figures 4b-f and 4d-h). In normal conditions, the non-
fluorescent calcein acetoxymethyl compound present
in live/dead assay transports into cells, and intracellular
esterases remove the acetoxymethyl group, thereby
producing strong green fluorescence. It is believed that
the borate ions present in the B3 glass interfere with the
c d acetoxymethyl group, thereby producing strong green
fluorescence even without cells. The PCL only scaffolds
do not exhibit strong fluorescence in the absence of glass
particles, which can be clearly observed in Figures 4a-e
and 4c-g (strong green fluorescence in Figure 4g is
because of more cells and not material). The background
staining makes it difficult to contrast and quantify cells
Figure 3. Scanning electron microscope images of near-field on PCL+B3 glass scaffolds using the ImageJ software.
electrospinning of polycaprolactone (NFES) and three-dimensional The green fluorescence and staining of the 3D printed
(3D) printed scaffolds soaked in complete culture media for 7 days.
Scaffolds showed HA-like crystal formations on the scaffold PCL+B3 glass scaffold filament can be clearly observed
surface. (a-b) NFES scaffold and its magnified image, (c-d) 3D in Figures 4b-f. Overall, the live/dead assay results
printed scaffold and its magnified image. qualitatively indicated more cell ASC proliferation in
NFES scaffolds compared to 3D printed scaffolds.
7 days. The assay reagents consist of a non-fluorescent To quantify cell proliferation, CyQuant assay was
calcein dye which is converted to green fluorescent calcein performed on the ASC-seeded scaffolds (Figure 5). The
after intracellular esterases in live cells causing the live CyQuant GR is a green fluorescence dye that intensifies
cells to stain green. In dying or dead cells, a bright red after it binds to the nucleic acid of DNA to provide a
fluorescence is generated on binding to DNA. Overall, reading that is then converted to cell number based on
in all scaffold types, the results indicated more live cells the standard curve. The results indicated increased cell
in comparison to dead cells (Figure 4). Specifically, more proliferation in NFES PCL scaffolds compared to 3D
live cells were observed in all NFES scaffolds with or printed PCL scaffolds after 1 day and 7 days incubation.
without glass in comparison to 3D printed scaffolds However, the results are not statistically significant
after 7 days incubation. This can be clearly seen in with P = 0.4. A wide pore size distribution in NFES
Figures 4g-h. However, the live/dead assay images were scaffolds could have provided uniform cell distribution
not used to quantify the cell viability results because of and proliferation, whereas cells were mainly observed
the high background staining of B3 glass particles, which on filaments in 3D printed scaffolds. The Live/Dead

4 International Journal of Bioprinting (2019)–Volume 5, Issue 1