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Wen Shing Leong, Shu Cheng Wu, Kee Woei Ng, et al.

Minisart High Flow 0.2 µm syringe filter unit was FTA 200 (First Ten Angstroms, USA) via the sessile
purchased from Sartorius Stedim Biotech S.A., Au- drop method with 0.5 μL of DI dispensed from the
bagne, France. Jung tissue freezing medium was pur- syringe of the system.
chased from Leica Instruments, Germany.
2.5 Mechanical Testing
2.2 Fabrication of Electrospun Scaffold
The mechanical properties of 2D electrospun scaffolds
Electrospun scaffolds were fabricated using electros- were investigated using Instron microtester 5848. The
pinning chamber, Nanon-01A (Mecc Co. Ltd, Japan). samples were die cut into dumbbell shapes according
Briefly, PCL was dissolved in DCM and DMF at to ASTM D638. The sample was tested under a
3.5:6.5 (v/v) ratio to obtain 13% (w/v) solution. The crosshead speed of 10 mm/min at room temperature.
polymer solution was spun through 21G metal nozzle 5 surface modified scaffolds and 5 surface non-mod-
at accelerating voltage 18kV and flow rate 0.5 mL/h. ified scaffolds were tested to investigate the effect of
Working distance was set at 15 cm for 2D scaffold surface modification on the mechanical properties of
collected on collector plate, and 7.5 cm for 3D scaf- the scaffolds.
fold collected on a 7.5 cm stainless steel medical hy-
podermic 18G needle insulated from ground. Elec- 2.6 SEM Characterization and Fiber Diameter
trospinning was carried out for 4 hours for collection Measurements
of 2D scaffold and 30 minutes for collection of 3D The electrospun PCL scaffolds were sputter coated
scaffold. Collected electrospun scaffolds were dried in with gold for 30 seconds at 18mA. The size and mor-
vacuum oven at 37°C for 1 week to remove any resi- phology of scaffolds were observed using SEM (JEOL
dual solvent. JSM-5310). Fiber diameters were measured using

2.3 Surface Modification ImageJ [28] from triplicates. At least 50 measurements
were taken at random locations in SEM micrograph.
Gelatin was grafted onto the electrospun fiber surface
through aminolysis [27] . Briefly, scaffolds were washed 2.7 Porosity and Pore Size Measurements
in 70% ethanol and deionized (DI) water, followed by Micrometritics Autopore IV 9500 Mercury Porosime-
immersion in 40%(v/v) ethylenediamine at room ter was used to investigate pore size and porosimetry
temperature for 14 hours. Scaffolds were then rinsed 3 of non-modified 3D and 2D electrospun scaffold. The
times with DI water for 10 minutes to remove free range of pore diameters, d p, could be calculated using
ethylenediamine. After that, scaffolds were immersed the Washburn equation (Equation (1)).
in 2.5% (by weight) glutaraldehyde (GA) for 4 hours
γ −
at room temperature, followed by washing 3 times d = 4 cosθ (1)
p
with DI water for 10 minutes. GA grafted scaffolds P
were then incubated in 3 mg/mL filtered gelatin at where γ is the surface tension of mercury, θ is the
37°C for 24 hours. Gelatin immobilized scaffolds contact angle between the mercury and the scaffold
were then rinsed 3 times with DI water for 10 minutes and P is the pressure. As reported in literature [29] , a
to remove free gelatin. Lastly, prior to cell culture, contact angle of 140° between PCL and mercury in air
scaffolds were sterilized under ultraviolet light for 10 was used. Four replicates were measured for each
minutes. scaffold type.
All chemical treatment and washing processes in-
volving the use of solution were carried out within 2.8 Cell Seeding
vacuum chamber to ensure complete perfusion of the HDFs were cultured in fibroblasts cell basal medium
solution. Prior to subsequent characterization, all (high glucose DMEM supplemented with 10% Gold
scaffolds were freeze-dried to ensure the volume and FBS, 1% penicillin streptomycin, 1% Amphotericin
shape of the scaffolds remained intact, and no mois- B). To prepare scaffolds for cell culture, 3D scaffolds
ture was trapped within the scaffolds. were cut into 0.6×0.6×0.6 cm cube while 2D scaf-
3
3
2.4 Contact Angle Measurement folds were cut into 1.6×1.7×0.08 cm sheet. The cells
3
were seeded at same density (500 cells/mm ) on 2D
Static water contact angle measurements on 2D elec- and 3D scaffolds. Cell culture media were changed
trospun scaffold membranes were carried out using every two days.
International Journal of Bioprinting (2016)–Volume 2, Issue 1 83