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International Journal of Bioprinting N-PLN hydrogels for human skin modeling

as a photoinitiator. Formulation 1 was freshly prepared previously silanized PET membranes. Immediately after
by dissolving the reagents in MilliQ water at room photocrosslinking, the samples were washed with PBS
temperature. In the case of Formulation 2, 1× conjugation to remove non-photocrosslinked residues and carefully
buffer (CB Buffer) was added. The pre-gel solutions were wiped with a Kimwipes® tissue (Kim Tech Science).
either photocrosslinked or stored at −20°C until the next Hydrogels were then kept in PBS at 37°C to induce
use. In the case of cell-laden constructs, a cell suspension swelling. Pictures of the hydrogels were taken at different
of Hs-27 human foreskin fibroblasts (5 × 10 cells/mL) was time points with an optical microscope (MZ10 F, Leica,
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prepared and mixed with the pre-gel solution right before Germany), and the scaffold dimensions were checked and
photocrosslinking (Figure 1b). analyzed using ImageJ software. The volumetric swelling
ratio was calculated as volume increase due to the swelling
2.3. 3D bioprinting system: Direct laser writing (difference between the swollen volume at a certain time
A custom-made 3D bioprinting system based on DLW “t” and the initial volume at time 0) and the initial volume
technique was employed to fabricate the hydrogel-based at time 0.
skin scaffolds. The apparatus is composed of (i) an optical
setup consisting of a 405 nm laser diode (DL5146-101S, 2.4.2. Rheology analysis
Thorlabs, NJ, USA), a collimator (LTN330-A, Thorlabs), For the rheology analysis, hydrogels were fabricated
a lens tube with a HR 10× objective with 0.45 of N/A by in situ crosslinking within the analysis apparatus.
(Edmund Optics, NJ, USA) coupled to a motorized DC A Discovery HR-2 rheometer equipped with an 8 mm
servo actuator (z-axis) (Z-series, Thorlabs), and (ii) a parallel Peltier plate and a UV light source (100–400 nm)
motorized translational stage, movable in x–y plane (Lightning cure LC8 Hamamatsu) was employed for this
TM
(Z-series, Thorlabs). All the adaptors and connectors are purpose. Drop volumes of 13 µL of pre-gel solutions were
also from Thorlabs. The system is connected to a personal crosslinked with 87.6 mW/cm power density, measured
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computer, which enables the control of the writing at the crosslinking plane. The storage (G’) and loss (G’’)
parameters, such as the speed and acceleration of the moduli were obtained as a function of the oscillation strain
translational stage, in both x and y directions, through for both pre-gel formulations, by applying a sweep of
LabView software. The laser beam parameters, like the focus amplitude between 0.01% and 10 % and keeping constant
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and the aperture, can be manually adjusted while the light the frequency to 1 Hz with a fixed running temperature
intensity is regulated by a power supply (LDC200C Series, of 25°C. Assuming a Poisson’s coefficient value for the soft
Thorlabs). Rectangular-shaped hydrogels (5 mm × 1 mm × hydrogels of 0.5, Young’s modulus (E) was calculated
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0.5 mm) can be crosslinked on a variety of supports, such from the values of the complex modulus (G*), derived
as well-plates, cover glasses, or PET membranes. For that from G’ and G’’ data.
purpose, the pre-gel solutions were exposed to light at a
power ranging from 1 to 41 mW for 10–15 s while the stage 2.5. Fabrication of cell-laden norbornene-pullulan
was translated in the x or y directions at a variable speed hydrogels
(0.15–0.30 mm/s). More complex in-plane geometries, for Human foreskin Hs-27 fibroblasts (ATCC-CRL 1634) were
example, grids and circles, can also be printed combining used in this study to mimic the dermal compartment of the
movements of both x and y motors at the same time. The skin-like constructs. Hs-27 were cultured and expanded
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working volume of the hydrogel solutions to be printed in 25 cm flasks in Dulbecco’s Modified Eagle’s Medium
was minimized by using circular PDMS pools (6 mm (DMEM; Gibco, Thermo Fischer Scientific, MA, USA),
in diameter and 0.5 mm in height), resulting in a 15 µL supplemented with 10% v/v fetal bovine serum (FBS;
solution per printed sample, proving the cost-effectiveness Gibco, Thermo Fischer Scientific) and 1% v/v penicillin/
of using biopolymers (Figures 1b and S1, Supporting streptomycin (Sigma-Aldrich, MA, USA). Hs-27 was
Information). Right after photopolymerization, samples
were washed with phosphate-buffered saline (PBS) and Table 1. Printing parameters employed to produce samples for
kept in submerged conditions at 4°C (without cells) or mechanical studies
37°C (with cells embedded) for later use.
Polymer formulation Power (mW) Stage translational
2.4. Mechanical characterization of norbornene- speed (mm/s)
pullulan hydrogels Formulation 1: 13 0.3
(N-PLN: PEG-link: LAP) 22 0.3
2.4.1. Volume swelling analysis 30 0.3
To characterize the swelling properties of the hydrogels, Formulation 2: 22 0.3
pre-gel solutions were irradiated at 405 nm wavelength (RGD-N-PLN: HA-MMP-
(Table 1) to photocrosslink hydrogels on top of link: LAP)

Volume 10 Issue 4 (2024) 225 doi: 10.36922/ijb.3395