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

particularly under physiological conditions (pH 7.4, natural origin and known biocompatibility of alginate and
37°C). 27,41,42 The hydrogel system exhibits rapid gelation, MC, along with laponite-RDS at non-toxic concentrations,
high yield stress, and prolonged stress relaxation, enabling support the formulation’s safety for tissue engineering.
efficient transitions between flow and solid states. These The consistent cell viability also suggests the stability of
properties contribute to reliable extrusion, improved the hydrogel under physiological ionic conditions. Thus,
print fidelity, and stable construct formation during 3D despite the presence of charged polymers such as GelMA,
bioprinting. MC, and alginate, no detrimental effects on cell viability

Collectively, these multiscale interactions, including were observed.
ionic bonding, hydrogen bonding, and polymer network We next characterized fibrotic responses of the
entanglement, synergistically regulate the rheological encapsulated keloid fibroblasts. Compared to normal
performance of the bioink. This cooperative behavior dermal fibroblasts, patient-derived keloid fibroblasts
enhances the pre-print stability and post-print mechanical showed significantly higher expression of ECM remodeling
integrity, enabling the formation of cell-laden constructs genes (COL1, MMP2, LOX), myofibroblast markers
with clinically relevant stiffness. Prior studies involving (ACTA2, Vimentin), the fibrosis-associated marker FSP1,
GelMA–clay–alginate systems have demonstrated similar and the inflammatory cytokine IL6 (Figure S3A and B). To
synergistic effects, 43-45 supporting the design rationale and assess the potential of the GxAxMxRx bioink for modeling
functional benefits of our composite formulation. fibrotic skin, we further examined gene expression

3.5. Biocompatibility of GxAxMxRx as a keloid responses to matrix stiffness. The stiffer G5A1M1R1
skin model formulation induced markedly higher expression of COL1
Keloids are fibrotic skin disorders characterized by (~90%), MMP2 (~70%), and IL6 (~85%) compared to the
excessive ECM deposition, particularly collagen types I, softer G4A1M1R1 formulation (Figure 6B), demonstrating
and abnormal fibroblast behavior, leading to stiff, nodular that even modest differences in stiffness can enhance the
lesions that extend beyond the original wound. A hallmark fibrotic and inflammatory phenotype of keloid fibroblasts
of keloid pathology is the activation of dermal fibroblasts in 3D culture. Additional upregulation of FSP1 (~18%) and
into myofibroblasts, marked by elevated expression of LOX (~20%) was also observed in G5A1M1R1, consistent
fibrosis-related proteins such as α-SMA and FSP-1. 18,46 with fibroblast activation and increased ECM crosslinking
To model this pathophysiology in vitro, we encapsulated activity. These findings are consistent with prior reports
patient-derived keloid dermal fibroblasts within two that increased matrix stiffness promotes mechanosensitive
GelMA-based bioink formulations: G4A1M1R1 (soft) signaling, fibroblast-to-myofibroblast differentiation, and
and G5A1M1R1 (stiff). These compositions represented excessive ECM remodeling, which are the hallmarks of
the lowest and highest stiffness values among all tested fibrotic tissue pathology. 18,48,49
hydrogels (Figure S1A), enabling us to assess the impact of To confirm these changes at the protein level, we
matrix stiffness on keloid fibroblast behavior. Importantly, performed immunofluorescence staining on single square-
the concentrations of MC, alginate, and laponite-RDS were shaped 3D-printed constructs (adapted from a 3 × 3
held constant across both groups to minimize compositional grid pattern; Figure S2A). Positive expression of MTS1
variability and isolate stiffness as the primary variable. The (FSP1), F-actin, and α-SMA was observed in encapsulated
selected formulations were specifically chosen to closely keloid fibroblasts (Figure 6C–D), with substantially
match the microscale elastic moduli of normal dermis stronger signal intensity and greater cell aggregation in
(~2.4 ± 1.0 kPa) and keloid tissue (~14.2 ± 1.0 kPa), as G5A1M1R1. These findings support the activation of
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previously reported. Thus, this design allowed us to myofibroblasts under stiff microenvironmental conditions.
mimic physiologically relevant mechanical cues and assess The transition of fibroblasts to myofibroblasts is a hallmark
mechanotransduction in a 3D-printed skin fibrosis model. of skin fibrosis, characterized by elevated expression of
Live/dead assays performed 3 days post-encapsulation α-SMA and FSP1, along with prominent actin stress fiber
showed strong calcein-AM fluorescence, confirming formation (F-actin). Together, these findings suggest that
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high cell survival within the 3D-printed constructs the GxAxMxRx bioink provides a mechanically tunable
(Figure 6A). These findings demonstrate the low cytotoxicity and biocompatible platform for constructing simplified 3D
and excellent biocompatibility of the GxAxMxRx hydrogel models that recapitulate key features of fibrotic skin tissue.
system. Notably, key functional motifs such as RGD By integrating GelMA, alginate, MC, and laponite-RDS,
sequences and MMP-sensitive linkages are preserved we developed a structurally stable scaffold that supports
during GelMA photopolymerization, facilitating cell cell viability and enables stiffness-dependent modulation
adhesion and matrix remodeling. Furthermore, the of fibrotic gene expression. While preliminary, this system
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Volume 11 Issue 4 (2025) 456 doi: 10.36922/IJB025160154