Page 235 - IJB-10-4

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

membranes). In this study, an EVOM3 Epithelial voltmeter sequence, which can be cleaved by a broad range of MMPs
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with an STX3 electrode (World Precision Instruments, and allows cells to locally degrade the polymer network if
Germany) was employed. Experimental TEER values they could produce the indicated MMPs. In the present
obtained were corrected by subtracting the resistances due formulation, the N-PLN polymer was also functionalized
to the hydrogels and the PET membranes and normalized with RGD peptide to enhance the scaffold’s performance
considering the surface area of the hydrogels. when cells were included.

2.7. Statistical analysis Initial printability tests showed defined gel geometries
Statistical analysis of data obtained from all the experiments using the following molarities for RGD: N-PLN: HA-
was performed. Data are presented as mean ± standard MMP-link: LAP = 0.92 mM: 2.5 mM: 2.5 mM: 0.5 mM,
error of mean (SEM) or standard deviation (SD). The with a molar ratio of polymer: crosslinker = 1:1, resulting
graphs were plotted using GraphPad Prism 8.3.0 software power intensity of 22 mW, and a stage translational speed
(GraphPad). Experiments for each working condition of 0.3 mm/s, which were the best printing parameters.
and formulation were conducted in two to six technical Upon determining the optimum molar content
replicates. The Student’s t-test was employed to analyze (polymer: crosslinker), the impact of printing parameters
the differences in mechanical characterization and level of was evaluated on the gels’ properties. The volumetric
markers. For cell viability, a two-way analysis of variance swelling ratio for Formulation 1 was first evaluated in
(ANOVA) was performed. samples crosslinked using a power range of 13–30 mW and
a stage translational speed of 0.3 mm/s (Table 1). For all the
3. Results and discussion polymerization conditions tested, the equilibrium swelling

3.1. Initial printability tests and physical properties was reached after 48 hours, showing an overall water
of norbornene-pullulan-based hydrogels uptake capacity of >10%, even though a peak of 27% was
Before the evaluation of the physical characteristics of reached for one specific crosslinking condition (22 mW,
N-PLN-based formulations, the initial light-induced 0.3 mm/s, Formulation 1), indicating that this intermediate
printability of the two different N-PLN-based formulations power may promote the water absorption capability of the
(Formulation 1 and Formulation 2) was assessed through network (Figure 2a).
DLW, to identify the proper crosslinking molar ratio The volumetric swelling behavior for Formulation 2
between the polymer, N-PLN, and the crosslinkers. First, was evaluated using samples photocrosslinked with only
N-PLN: PEG-link: LAP solution (Formulation 1) was a power intensity of 22 mW and a stage translational
explored in this respect. Some studies have pointed out speed of 0.3 mm/s (previously optimized for the specific
that pullulan and PEG are not miscible in certain ratios molar ratio used), also considering the highest uptake
due to phase separation. 35,36 Upon visible light irradiation, capacity registered for Formulation 1 with these printing
the pre-gel solution containing 5 mM N-PLN, 4 mM PEG- parameters. In this case, the equilibrium swelling ratio was
link, and 0.2 mM LAP successfully forms a well-defined ~20%, showing less capacity of these gels to retain water
crosslinked network, resulting in a shape-retaining scaffold within the scaffold (right column in Figure 2a), compared
with good consistency and amenability to easy handling. to Formulation 1, and simultaneously suggesting a more
This performance surpasses that of other evaluated molar compact network.
ratios (5:1, 5:2, 5:3 for N-PLN: PEG-link), when using
the same printing parameters (power intensity of 22 mW The swelling is an important parameter to consider
and a stage translational speed of 0.3 mm/s) (see Figure since it depends on the crosslinking degree and the
S2, Supporting Information). This superiority may be porosity, having an impact on the bulk geometry, mass
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attributed to the optimal balance achieved between the transfer, and therapeutic delivery of substances. 39–41
functional thiol (SH-) groups present in both the polymer The greater the extent of crosslinking, the less flexible a
and the linker. As expected, a decrease in the molar ratio hydrogel is to change phase, swell, or shrink, in response
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between both species correlates with an increase in the to stimuli. Therefore, the choice of materials that exhibit
crosslinking rate, with the 5:4 ratio yielding the most this property is fundamental. It was reported that pure
favorable gelation results. collagen-based gels did not show swelling; in contrast with
chemically crosslinked gels such as collagen-HA or thiol-
Later, a hyaluronic acid-based crosslinker (HA-MMP- 43
link) was also examined using RGD-N-PLN: HA-MMP modified HA-based gels.
Link: LAP solution (Formulation 2). This formulation The bulk mechanical behavior of the two different
contains a thiol-modified-HA crosslinker coupled with formulations was also investigated. To this end, rheology
a matrix metalloproteinase (MMP)-cleavable peptide measurements were conducted to monitor the crosslinking

Volume 10 Issue 4 (2024) 227 doi: 10.36922/ijb.3395