Page 232 - IJB-10-4

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

commonly used radical-mediated polymerization, the incubated for 1–2 h on ice (while shaking occasionally) or
thiol-ene click reaction is faster, employs less photoinitiator at 4°C overnight, followed by an additional gentle mixing.
concentrations, and is not inhibited by oxygen, resulting RGD-N-PLN was provided in a solution containing 2.8
in hydrogel network with a highly ordered structure. 28–30 mmol/L RGD peptide and 10 mmol/L norbornene groups.
Here, norbornene-pullulan (N-PLN) formulations A conjugation buffer (3D-Life 10× CB Buffer, pH 7.2;
combined with different crosslinkers were successfully B20-3, Cellendes GmbH, Germany) was also included in
employed as a bioink in a customized direct laser writing the formulation with RGD-N-PLN. Lithium phenyl (2, 4,
(DLW) set-up to produce epithelized dermal skin 6-trimethyl- benzoyl) phosphinate (LAP) (TCI Chemicals)
constructs. The dermal compartment was created through was used as a photoinitiator.
the photocrosslinking of a pre-gel solution containing
human fibroblasts, while the epidermal compartment 2.2. Preparation of norbornene-pullulan
was developed by seeding human keratinocytes on top pre-gel solutions
of the fibroblast-laden hydrogels. As shown in this study, Two different pre-gel solutions based on N-PLN were
fibroblasts could survive, elongate, and spread within the prepared: (i) N-PLN in combination with a thiol-
N-PLN matrix up to 24 days post-encapsulation, without conjugated PEG-link (Formulation 1, containing N-PLN,
showing matrix contraction or collapse due to the cells’ PEG-link, and LAP), and (ii) RGD-N-PLN in combination
presence. Immunostaining studies revealed the expression with HA-based linker, HA-MMP-link (Formulation 2,
of basement membrane proteins and keratinocyte surface containing RGD-N-PLN, HA-MMP-link, and LAP).
colonization, consistently characteristic of an epidermal Orthogonal thiol-ene click chemistry was selected as
compartment, thus indicating the successful recapitulation an alternative to the homopolymerization reactions of
of a skin-like structure. This approach, combining visible- acrylates or methacrylates for the fabrication of hydrogel-
light photopolymerization and N-PLN -based materials, based scaffolds due to its extremely fast photocrosslinking
presents a promising method for the fabrication of human reaction, resulting in homogeneous structures with
skin constructs, which is readily employable in in vitro drug a high degree of control over the number of reacted
evaluation for pharmacological and toxicological testing. functionalities. 31,32 Once it starts, the radical step-growth
reaction advances in a stochiometric ratio until the limited
2. Materials and methods moiety (thiol or norbornene) is depleted or unavailable
(Figure 1a). As a result, the gelation can be achieved
2.1. Materials within seconds, with a low concentration of photoinitiator
N-PLN, RGD-modified N-PLN (RGD-N-PLN), thiol- or macromer. 31
modified polyethylene glycol (PEG-link; 3-D Life PEG
Link, L50-1), and thiolated HA modified with a matrix To create the scaffold, a pre-gel solution was prepared
metalloproteinase (MMP)-cleavable peptide (HA-MMP- (~150 µL) by mixing the combination of polymer and
link) were obtained from Cellendes GmbH, Germany. The crosslinker (Formulation 1—N-PLN: PEG-link: LAP
HA-MMP-link lyophilisate was dissolved in Milli-Q water = 5 mM: 4 mM: 0.2 mM; and Formulation 2—RGD-
to obtain a concentration of 10 mmol/L thiol groups. The N-PLN: HA-MMP-link: LAP = 0.92 mM: 2.5 mM: 2.5
freshly dissolved lyophilisate was gently vortexed and mM: 0.5 mM) in MilliQ water, in the presence of LAP

Figure 1. Photopolymerization process of norbornene-based hydrogels. (a) Reaction mechanism of norbornene-based thiol-ene click chemistry.
Reprinted with permission of John Wiley and Sons, Copyright © 2024 Wiley Inc. (b) Schematic illustration of hydrogel photopolymerization with Hs-27
31
encapsulation and consequent mounting within Transwell inserts. Scale bars = 1 mm.
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Volume 10 Issue 4 (2024) 224 doi: 10.36922/ijb.3395