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Yang, et al.
inks were determined along with the testing time up to the fluorescent-labeled rhCol3. The rhCol3-contained
900 s under a constant temperature of 37°C, while the hydrogels were then prepared and incubated in PBS.
shear strain and frequency were maintained at 0.5% The 500 μL PBS was collected from each sample and
and 1 Hz, respectively. The time taken for the initial the same amount of fresh PBS was added. The time to
storage modulus of each bioink group to reach 50% collect the PBS was set to 1 h, 3 h, 5 h, 10 h, 1 d, 3 d,
and 80% of the averaged storage modulus at the post- 5 d, 10 d, and 14 d after initial incubation in a 37°C
UV crosslinking stage was also determined to illustrate incubator. The fluorescent intensity was determined by
the photo crosslinking efficiency. a microplate reader (Molecular Devices, SpectraMax
To evaluate the effect of GelMA and rhCol3 M2) at excitation wavelength of 494 nm and emission
concentration on the mechanical properties of the gelled wavelength of 518 nm.
bioinks, the compressive modulus of the hydrogels was
determined by compression test in this study. The square- 2.10. Engineering human skin equivalent in
shaped samples with 14.5 mm side length and 4.5 mm transwell inserts
height were prepared and compressed up to 50% strain at
the rate of 2 mm/min using a mechanical test instrument To create human dermal constructs in transwell inserts,
(Bose ElectroForce 3200, Bose Corp.). The compressive the line distance of the model was adjusted to 0.1 mm,
modulus was determined from the initial linear slope and the printing shape was changed to round to adapt
6
(10 – 15% strain) of the stress-strain curves. the 24-well transwell. Bioinks that contained 1 × 10 /mL
HDFs were prepared and then transferred into a 3 mL
2.8. Printability characterization syringe equipped with a 25G extrusion needle before
printing. A 405 nm UV light was used to crosslink the
The printability was assessed by a filament fusion test construct immediately after printing. A 200 μL fresh
consisting of printing parallel strands at decreasing medium was added into the transwell insert followed by
spacing, according to a previous study . The initial placing the transwell into a 24-well plate that contained
[30]
filament distance was set to 2.5 mm, decreased 0.1 mm 500 μL medium. The printed dermal layer constructs were
for each subsequent line, and ended at the distance of
0.3 mm. The printing head and the collecting platform cultured for 3 days before seeding the HaCaTs on their
2
5
of the bioprinter (Biomaker 2, SunP Biotech) were upper surface at a density of 2 × 10 cells/cm . They were
set to 23 and 10°C, respectively. The printing speed of cultured with submerged medium for another 5 days,
the nozzle was 2 mm/s, and the extrusion speed was followed by switching to the ALI medium while lifting
0.3 mm /s; a 25G extrusion needle was selected in the the insert to a level where the HaCaTs contact the air,
3
test. The printed parallel strands were cross-linked by a allowing for an ALI culture condition for HaCaTs. Both
405 nm light source for 20 s to form gelled construct and the submerged medium and ALI medium were changed
photos recorded by a digital camera. The fused filament every other day. The attached areas of proliferated
length (fs) at each edge of the meandering pattern to their HaCaTs were imaged with microscopy at days 3 and 6
corresponded filament thickness (ft) was determined and until the cells reached full confluence and the percentage
plotted for each filament distance (fd). Lattice structures of the cover area was determined by Matrix Laboratory
were also printed out to assess the printability (Pr value) (MATLAB R2019a). LIVE/DEAD™ staining assay was
of the bioinks based on a well-established approach . also applied to identify the proliferation of the HaCaTs on
[31]
Pr denotes value of printability that is defined based on dermal surfaces.
square shape using the following equation: Pr=L /16A, 2.11. Skin equivalent characterizations
2
where L and A denote the perimeter and the area of the
square, respectively. The printed skin constructs were harvested from the
transwell insert at week 3 and week 6 since the initial
2.9. rhCol3 dissolution test culture or ALI culture. The skin constructs were

To quantify the releasing of the rhCol3 from the gelled fixed with 4% paraformaldehyde for 20 min at room
bioinks, rhCol3 was conjugated with fluorescent dye. temperature before paraffin embedding. They were then
Predetermined rhCol3 and NHS-fluorescein were fully cut into sections and mounted onto slides for hematoxylin
dissolved in PBS to prepare the 10 wt% and 0.6 wt% and eosin (H&E) staining. For immunohistochemistry
solutions, respectively. The prepared solutions were staining, the sections were deparaffinized and rehydrated
then mixed and stirred at 37°C for 3 h in dark room. The with xylene and ethanol and were immersed in EDTA
well-mixed solution was then diluted with four volumes antigen retrieval buffer, followed by blocking with 3%
of PBS and transferred to dialysis bag (MW.14000) to BSA for 30 min. The slides were then incubated overnight
dialyze the unreacted fluorescent dye for 14 days. The at 4°C in a humid chamber, with primary antibodies
final dialyzed solution was then freeze-dried to obtain against cytokeratin 14 (1:100, Abcam) and cytokeratin

International Journal of Bioprinting (2022)–Volume 8, Issue 4 149