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Hou, et al.
pellets and reinforcements were heated at 150°C the scaffolds were calculated using the software
and mixed together in a crucible for 30 min. The Origin (OriginLab, USA).
material was stirred for 30 min to disperse the
fillers homogenously. After cooling down for 3 h, 2.4 Surface wettability evaluation
the mixed materials were then cut into pellets and Apparent water-in-air contact angle (WCA) tests
prepared for printing. Scaffolds were produced were carried out with a KSV CAM 200 system
using the screw-assisted material extrusion (KSV Instruments, Finland) to evaluate the surface
additive manufacturing system 3D DiscoveryTM hydrophilicity of the scaffolds. 2 ml of distilled
Evolution Bench-top (regenHU, Switzerland). water was dropped on the surface of the scaffold
The following design and processing parameters using a micrometric liquid dispenser (Hamilton,
were considered: Fiber layout of 0°/90°, melt USA) and the drop shape was recorded with
temperature of 90°C, screw rotation rate of 8 rpm, a DMK 21F04 FireWire monochrome camera
deposition velocity of 13 mm/s, slice thickness of (Imaging Source, Germany). Attention Theta
270 μm, and pressure of 6 bars. These parameters software (Biolin Scientific, Sweden) automatically
are considered to guarantee constant filament calculates the contact angle.
diameter of 330 μm after printing.
2.5 Biological studies
2.2 Morphological evaluation
In vitro, biological studies were conducted using
Morphological characterization was performed hADSCs (Invitrogen, USA). hADSCs were
through scanning electron microscopy (SEM). defrosted and cultured in MesenPRO RS™ basal
FEI Quanta 250 SEM (Thermo Fisher Scientific, medium (Thermo Fisher Scientific, USA) in
USA) was used to capture images of both the top T75 cell culture flasks (Sigma-Aldrich, UK) at
surface and cross-section of the scaffolds, using standard conditions (37°C, 5% CO concentration,
2
an accelerating voltage of 10 kV. Before image and 95% humidity) in a New Brunswick Galaxy
®
capturing, the scaffolds were cut into 3 mm blocks 170 R incubator (Eppendorf, USA). Cells were
and coated with a thin layer of metal (10 nm harvested at approximate 80% confluence using
gold) using the EMITECH K550X sputter coater 0.05% trypsin-EDTA (Invitrogen, USA) and
(Quorum Technologies, UK). The obtained images Centra MP4 Refrigerated Centrifuge (Thermo
®
were processed by ImageJ (NIH, USA). Fisher Scientific, USA) (1200 rpm, 150 s) before
2.3 Mechanical evaluation cell seeding. Cells ranging from passage 2 to 6
were considered for biological evaluation.
To determine the mechanical properties of the Before cell seeding, scaffolds were sterilized in
scaffolds, according to American Society for 99.8% ethanol (Thermo Fisher Scientific, USA)
Testing and Materials standards [31,32] , uniaxial for 4 h, transferred to a 24-well plate, and rinsed
compression tests were conducted on the with Dulbecco’s Phosphate-Buffered Saline (PBS)
INSTRON X testing system (High Wycombe, (Thermo Fisher Scientific, USA). 50000 cells
UK) with a 100 N load cell. The scaffolds were (counted by Cellometer Auto 1000 Bright Field
cut into blocks of 3 mm of width, 3 mm of length, Cell Counter [Nexcelom Bioscience, USA]) in
and 4mm of height. Compression tests were 0.8 mL medium were seeded on each scaffold
performed in dry state with the strain ranging sample and empty well (control group).
from 0 to 0.3 mm/mm (30%) and a displacement The viability and proliferation of cells
rate of 0.5 mm/min. Force F and corresponding were evaluated at 1, 3 and 7 days after cell
displacement ∆h were measured by sensors seeding using the Alamar Blue assay, which can
while the samples were compressed, and then quantitatively monitor the metabolic activity of
transformed into stress σ and strain ε values. The cells and potential cytotoxicity of scaffolds .
[33]
compressive modulus and compressive strength of At each particular time point, 1 mL of medium

International Journal of Bioprinting (2020)–Volume 6, Issue 2 63