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Kolan KCR, et al.
attachment, scaffolds were transferred to 35 mm Petri In this study, one of our aims was also to compare and
dishes with 2 ml of CCM. A Live/Dead cell imaging contrast the NFES scaffolds with 3D printing scaffolds
kit (ref. R37601, Eugene, OR) was used for qualitative in terms of bioactivity and cell proliferation. Therefore,
assessment of cell viability. Briefly, after a 24 h incubation we have used the solvent-based 3D printing process
period, scaffolds were washed with PBS, stained for to fabricate scaffolds with the same compositions of
30 min at room temperature, and examined under a PCL+B3 glass and PCL pastes used in NFES technique.
fluorescent microscope (Olympus IX51, Melville, NY). More details about the solvent-based 3D printing and
Five scaffolds were examined per experimental group, scaffold fabrication with PCL+B3 glass composite (up to
with at least five pictures taken per scaffold. 50 wt.% glass) can be found in our previous work [14,15] .
To quantify cell viability, scaffolds were analyzed 3D printed scaffolds were designed to have pore sizes
for total DNA using CyQuant cell proliferation assay not exceeding the pore sizes obtained using NFES
(Invitrogen), using the manufacturer’s protocol to technique (i.e., 250 µm) and to reflect an average pore
normalize all results to cell number. Briefly, 24 h after size. A printing speed of 20 mm/s, air pressure of 30 psi,
seeding cells, cellularized scaffolds were gently washed and filament-to-filament spacing of 0.35 mm were used
with PBS and frozen at −80°C overnight. Scaffolds to fabricate the scaffold. The average filament width of
were thawed the next day and analyzed with CyQuant. 167 ± 38 µm and pore size of 188 ± 28 µm were obtained
A sample size of n = 5 was used for all experiments for the 3D printed scaffold.
except for CyQuant assay for 3D printed PCL scaffolds
(n = 4). Scaffolds without cells were used for background 3.2. Scaffold Bioactivity
controls. One-way ANOVA was performed in Minitab The scaffold porosity was calculated based on 2D optical
to analyze the results and difference was considered images after the printing of the first two layers of the
significant if P < 0.05. scaffold with both processes. The obtained porosity for

3. Results and Discussion NFES scaffold was ~50% compared to ~30% for 3D
printed scaffold. The higher porosity and wide range of
pore sizes of NFES scaffold are beneficial for B3 glass
3.1. Scaffold Fabrication dissolution because of the larger surface area. Scaffolds

The effect of fabrication parameters including applied were soaked in CCM for up to 7 days to evaluate
electric field, printing speed, and extrusion pressure on the formation of hydroxyapatite (HA)-like layer on
the filament deposition was investigated. The above the scaffold surface (Figure 3). The results indicated
parameters control the porosity of the fabricated part by nanosized HA-like crystal formations on scaffolds made
changing the filament size, fiber size, and the amount of by both processes. The crystals were uniformly spread out
deposited material as shown in Figure 2a. The filament on NFES scaffold surface and were observed in patches
and fiber sizes at different parameters are shown in on 3D printed scaffolds. This indicates a faster B3 glass
Table 1. A 10 kV/cm electric field with 5 mm/s printing dissolution from NFES scaffolds compared to 3D printed
speed and 30 psi extrusion pressure (test #8) provided scaffolds. The X-ray diffraction results showed peaks
a suitable filament with randomly distributed small indicating the formation of non-stoichiometric HA which
fibers to create a biomimetic 3D structure. The printing is consistent with our previous studies where PCL+B3
[14]
schema and fabricated 3D scaffold with cancellous bone glass scaffolds showed similar conversion .
microstructure similarities can be observed in Figures 2f 3.3. Cell Viability and Proliferation
and 2g. Furthermore, the pore size distribution in NFES
scaffolds varied from 20 µm to 250 µm that are desirable Viability of ASCs was studied by seeding cells on
for bone tissue growth. scaffolds and performing live/dead assay after 1 day and

Table 1. Effects of fabrication parameter on filament and fiber sizes in NFES
Test Electric field (kV/cm) Printing speed (mm/s) Extrusion pressure (psi) Filament size (μm) Fiber size (μm)
#1 12 7.5 50 565±37.4 80.2±12.1
#2 10 7.5 50 567.5±37.9 46.8±6.9
#3 8 7.5 50 562.5±52.6 87.2±20.8
#4 6 7.5 50 N/A 103±10.9
#5 10 5 50 578.75±41.9 47.8±18.5
#6 10 2.5 50 501±28.6 99.8±28.8
#7 10 5 40 350.25±87.2 42.2±18.5
#8 10 5 30 517.5±27.9 33.4±6.3
#9 10 5 20 N/A 48±12.0

International Journal of Bioprinting (2019)–Volume 5, Issue 1 3