Page 501 - IJB-10-3

International Journal of Bioprinting 3D printing microgroove nerve conduits

The differential scanning calorimetry (DSC) test, rinsing with PBS, trypsinising (0.25% Trypsin-EDTA), and
which analyses thermal behaviour and crystallisation of neutralising with fresh media.
samples, was performed using TA Instruments Q-100 The microgroove films (3 wt% concentration and 20
(TA Instruments, New Castle, USA) from 0 to 200°C at µm thickness) were fixed in CellCrown inserts (Sigma-
TM
a heating rate of 10°C/min in a nitrogen atmosphere. Aldrich, UK) within 48-well plates to prevent them from
The degree of crystallinity (X ) and melting point (T ) of floating, sterilised with ethanol for 4 h, and washed twice
m
c
PCL and PLA were determined. X was calculated using with PBS. Cells were counted (Cellometer Auto 1000,
c
Equation I: Nexcelom Bioscience, USA), and films were seeded with
4
Χ (%) = ΔH /(ƒ×ΔH ) × 100 (I) 1.9 × 10 cells in 0.1 mL of cell culture medium specifically
0
m
c
on the surface. After incubating for 30 min to allow the
where ΔH is the specific melting enthalpy of the cells to attach, 0.4 mL of cell culture medium was added.
m
sample, ƒ is the weight percentage of PCL or PLA, The media was refreshed every 3 days.
and ΔH is the melting enthalpy of completely crystalline 2.9. Cell viability, proliferation, and morphology
0
polymer (132 J/g for PCL, 93.7 J/g for PLA). 47 The Alamar Blue assay was used to monitor cell metabolic
2.6. Tensile mechanical assessment activity, which serves as an indicator of cell viability and
Tensile tests were conducted with the Instron 3344 (Instron, proliferation. Following the manufacturer’s instructions,
USA) and integrated software (Bluehill Universal, Instron, briefly, 50 µL of Alamar Blue solution was added to each
USA). The samples (n = 5), each measuring 1.5 mm and 10 well and incubated for 4 h at 1, 3, 5, and 7 days after cell
seeding, and 150 µL of the solution was transferred to 96-
mm, for width and length, respectively, were prepared. A well plate (n = 5). The fluorescence (ex 530 nm/em 590
crosshead speed of 1 mm/min, 6 mm grip distance, and a nm) was measured by a microplate reader (Synergy HT,
10 N load cell were used with the force applied in parallel BioTec, USA). The well plate was changed every time after
to the direction of the grooves. The samples were prepared Alamar Blue assay.
with different thickness (10, 20, or 30 µm), different
solvent concentration (1, 3, or 5 wt%), and different groove Cell viability was observed on day 7 using a live/dead kit
morphologies. The maximum tensile strength, the strain and following the manufacturer’s instructions. Calcein-AM
at breaking, and the Young’s modulus were analysed using (green) and ethidium homodimer-1 (EthD-1) (red) were
Origin software (OriginLab, USA). used to fluorescently label live and dead cells, respectively.
Samples (n = 3) were washed three times with PBS before
2.7. Surface wettability staining in a calcein-AM (1/2000 dilution) and EthD-1
The wettability of the microgroove films was assessed by (1/500 dilution) PBS solution and incubated for 20 min
water contact angle (WCA) measurement. The microgroove prior to imaging with a confocal microscope (Lecia SP-8
thin films with different groove sizes, different materials, Lightning, Lecia Microsystems, Germany). A minimum of
and different solvent concentrations were prepared for the three images per sample and a minimum of 180 cells in
WCA to examine the hydrophilicity. The wetting behaviour total were used to calculate the percentage viability.
of the films (n = 5) was evaluated using a DSA100B (KRÜSS, Cell morphology was observed on day 7. The samples (n
Germany) with an image taken at 0 s and 60 s after droplet = 3) were rinsed twice with PBS (37°C), fixed with 4% PFA
formation on the sample surface (static sessile drop). The for 15 min, washed with PBS three times, permeabilised
contact angle was automatically calculated by Attention with 0.1% Triton X-100 for 15 min at room temperature,
Theta software (Biolin Scientific, Sweden) through the and washed with PBS three times. A 5% FBS solution was
sessile drop and drop profile fitting method. then added to block non-specific binding, and the samples
were incubated for 1 h and washed once with PBS. Alexa
2.8. In vitro cell culture Fluor® 488 Phalloidin (1:400) in 1% FBS solution was
The SH-SY5Y neuroblastoma cell line (ATCC® CRL- added to each sample and incubated for 45 min at room
2266) is a well-established in vitro model for neuronal temperature and protected from light. The samples were
differentiation and neurotoxicity. SH-SY5Y cells were rinsed in PBS, and DAPI (1:1000) was added and incubated
48
cultured in T75 cell culture flasks (Sigma-Aldrich, UK) with for 10 min at room temperature in the dark. The samples
DMEM/F-12 (1:1, v:v) medium supplemented with 10% were washed twice with PBS and stored at 4°C prior to
foetal bovine serum (FBS), 1% penicillin/streptomycin, imaging. Cell morphology was visualised with confocal
and 1% glutamine, in an incubator at 37°C and 5% CO . microscope (Leica SP8 Lightning, Leica Microsystems,
2
The growth medium was changed every 3 days. Cells Germany), and cell orientation was quantitatively analysed
were sub-cultured after reaching 80–90% confluence by by ImageJ software. 46

Volume 10 Issue 3 (2024) 493 doi: 10.36922/ijb.2725