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International Journal of Bioprinting Tunable GelMA-based bioinks for keloid modeling

Ray XX-15L; UVP, USA) for 5 min for crosslinking. The 2.9. Statistical analysis
hydrogel was kept in the growth medium for 3 days before Data were analyzed using GraphPad Prism version 10.3
further experiments. (GraphPad Software, Inc., USA). Bar graphs represent mean
values, with error bars indicating standard deviations. The
2.6. Live/dead cell viability assay statistical significance between two groups was assessed
Cell viability was assessed three days after the printing using Student’s t-test, while comparisons among multiple
process using the Live/Dead Viability/Cytotoxicity groups were conducted using one-way analysis of variance
Kit (cat# 3 L-3224, Invitrogen, USA), following the followed by Tukey’s post hoc test. Statistical significance
manufacturer’s instructions. After staining, the hydrogel was set at *p < 0.05, **p < 0.01, and ***p < 0.001.
was washed with PBS and imaged using a THUNDER
Imager (Leica Microsystem, Germany) with 5× objective 3. Results and discussion
lens (N Plan; NA, 0.12; Cat# 11506303, Leica Microsystem, 3.1. GelMA concentration as a
Germany) and an EVOS M700 imaging system (Thermo biocompatible crosslinker
Fisher Scientific, Waltham, USA) equipped with a 10× The GxAxMxRx bioink system was formulated through
water immersion objective lens (Fluorite; NA, 0.30), polyelectrolyte complexation and covalent crosslinking
utilizing a stitching function to generate complete images among GelMA (G), alginate (A), MC (M), and laponite-
of the hydrogel. RDS (R), as illustrated in Figure 1. The variable “x”
2.7. Real-time polymerase chain reaction denotes the respective concentrations used (Table S1).
Total RNA was isolated from the hydrogel using TRIzol Each component serves a distinct role: GelMA acts as a
reagent (Cat# 15596018, Ambion, USA), and cDNA synthesis biocompatible crosslinker offering structural integrity and
was performed using ReverTra Ace qPCR RT Master Mix cell-adhesive moieties, while alginate, MC, and laponite-
with gDNA Remover (Cat# KMM-101, TOYOBO, Japan), RDS modulate viscoelasticity and printability.
following the manufacturer’s instructions. Subsequently, GelMA is an ampholytic polymer carrying both
qPCR was performed using a QuantStudio 1 Real-Time negative and positive charges. At acidic pH values below its
PCR System (Applied Biosystems, Foster City, USA) and isoelectric point, its amine groups are protonated, rendering
SYBR Green Real-time PCR Master Mix (Cat# 4472918, it positively charged and enabling electrostatic interactions
TOYOBO, Japan). Table S3 lists the primers used for the with the negatively charged MC to form polyelectrolyte
24
qPCR. All experiments were performed in triplicate for complexes. Concurrently, alginate contributes to the
each group. Glyceraldehyde 3-phosphate dehydrogenase polymer network through nucleophilic attack by its
(GAPDH) served as a housekeeping gene. The ΔC values carboxylic acid groups on the hydroxyl functionalities
t
were determined as Ct target − Ct GAPDH , and relative fold of MC, leading to ring-opening of the pyranose
25
changes were calculated using the 2 −ΔΔCt method. 23 structure and formation of aldehyde intermediates.
These intermediates react with GelMA’s amine groups,
2.8. Immunofluorescence generating nitrogen-containing intermediate rings.
In situ hydrogels were fixed in 4% paraformaldehyde (PFA, Additionally, primary amines on GelMA can form stable
Cat# CNP015-0500, CellNest, Korea) for 30 min and amide bonds with carboxylic or ester groups on alginate,
permeabilized with 0.3% Triton X-100 (Cat# TRX777.500, enhancing both intra- and inter-molecular crosslinking
Bioshop, Canada) solution for 30 min at room temperature. and improving hydrogel stability. Furthermore, alginate
26
Subsequently, hydrogels were blocked in a solution of 5% and MC primarily interact with GelMA through physical
BSA for 30 min at room temperature. Hydrogels were then entanglement and hydrogen bonding, which increase
incubated in primary antibodies for 48 h at 4°C, followed viscosity and enhance viscoelasticity. These interactions
by secondary antibodies for 24h at 4°C. Antibodies are also promote shear-thinning behavior, thereby improving
listed in Table S4. Confocal imaging was performed the printability of the hydrogel system. 27
using an LSM 710 microscope (Carl Zeiss, Germany) at Laponite-RDS, a synthetic nanoclay composed of disc-
the Soonchunhyang Biomedical Research Core Facility like platelets (~15 nm diameter, ~1 nm thickness), features
of Korea Basic Science Institute with a 10× objective lens alternating trioctahedral and tetrahedral layers with
(Plan-Apochromat; NA, 0.45; Cat# 420640-9900-000, interspersed magnesium and lithium ions surrounded
Carl Zeiss, Germany). Tiled images were acquired using a by negatively charged silicate surfaces. This high charge
THUNDER Imager (Leica Microsystem, Germany) with a density and surface area render it suitable for biomedical
5× objective lens (N Plan; NA, 0.12; Cat# 11506303, Leica applications. In bioink formulations, laponite-RDS
Microsystem, Germany). enhances mechanical stiffness, shear-thinning behavior,

Volume 11 Issue 4 (2025) 449 doi: 10.36922/IJB025160154