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International Journal of Bioprinting Dual tuning of 3D-printed SilMA hydrogel

paraffin, and sectioned at 5 μm. The sections were As shown in Figure 1C, both SilMA and PEO/SilMA
stained with HE, Safranin O, and Alcian Blue, and groups exhibited acceptable 3D printing performances.
immunohistochemically stained for collagen II (Col- The PEO/SilMA combination yielded translucent matte-
II) and collagen I (Col-I). The positive area and average white hydrogels, while NFs-incorporated groups exhibited
optical density values of Col-II and Col-I were statistically opaque white coloration with significantly enhanced
analyzed through Image J software (version 1.51j8). printability, consistent with previous findings. 30–32
While nanomaterials enhanced hydrogel formability
2.9. Statistical analysis and mechanical strength in 3D bioprinting, it was
All data were expressed as mean ± standard deviation. The noticeable that excessive nanomaterial concentrations
data were analyzed using one-way analysis of variance with might compromise mechanical reinforcement. The
GraphPad Prism version 9.5.1, and p < 0.05 was considered sample with 3% NFs could not be formed during the 3D
statistically significant. printing in this study. Regarding the concentration of
3. Results and discussion PEO, initial trials using a 1:1 volumetric ratio of 2%NF/
SilMA and PEO emulsion produced unprintable hydrogels
3.1. Preparation of silk methacryloyl hydrogels and (Figure S2). Therefore, a revised 2:1 mixing ratio was
electrospun silk fibroin nanofibers selected. To evaluate potential PEO residue in hydrogels
Figure 1A displays the H-NMR spectra of the prepared after immersion-induced precipitation, SilMA and PEO/
1
SF and SilMA. Notably, the presence of vinyl methacrylate SilMA hydrogels soaked for 2 days were lyophilized and
signals at δ = 6.2–6.0 parts per million (ppm) and δ = 5.8– analyzed using FTIR spectroscopy (Figure 1B). The near-
5.6 ppm in the spectrum of SilMA, along with the methyl identical FTIR spectra of both hydrogel groups confirmed
group signal at δ = 1.8 ppm and a gradual decrease in the complete PEO removal after the 2-day immersion. This
lysine methylene signal at δ = 2.9 ppm, suggests that the outcome establishes a biocompatible foundation for cell-
lysine residues in SF underwent chemical modification. laden hydrogel fabrication using PEO-based assembly.
Additionally, a detectable signal at δ = 3.2–3.6 ppm was
observed, which is attributed to hydrogen in the adjacent 3.3. Characterization of nanofiber-enhanced porous
carbon–nitrogen bond. In the FTIR spectrum of SF, the silk methacryloyl hydrogels
characteristic β-sheet amide I, amide II, and amide III 3.3.1. Morphology and porosity
bands were identified. For SilMA, in addition to these Figure 2A schematically illustrates the internal structures
β-sheet features, a weak absorption band at 1297 cm was of SilMA, PEO/SilMA, and PEO/NF/SilMA hydrogels.
−1
observed, corresponding to the CHOH group stretching Optical microscopy revealed PEO emulsion droplets
from the alcohol group in GMA following the epoxy ring within the matrix to characterize the NF-enhanced
opening. Furthermore, a change at 1118 cm was also porous hydrogels. Due to the reduced light transmittance
−1
detected, corresponding to methylene rocking vibrations in hydrogels after NF incorporation, SilMA, and PEO/
of the vinyl methacrylate group in GMA (Figure 1B). SilMA hydrogels were selected for structural observation.
20
Collectively, FTIR and H-NMR spectra demonstrated As shown in Figure 2B, the SilMA group contained small
1
the successful extraction and methacrylation of SF.
As demonstrated in Figure S1A, the SilMA solution droplets formed through self-cross-linking, while the PEO/
containing LAP transitioned from a liquid to an immobile SilMA group exhibited significantly larger near-spherical
gel state under UV irradiation. Additionally, SEM images PEO emulsion droplets, confirming the pore-forming effect
(Figure S1B) revealed that the electrospun SF NFs exhibited of PEO phase separation. The hydrogel microstructure
uniform, smooth morphology, and nanoscale diameters, was characterized using SEM, rhodamine B staining, and
confirming successful SF NF fabrication. HE staining (Figure 2C). SEM images revealed a well-
defined porous architecture in the PEO-incorporated
3.2. Preparation of nanofiber-enhanced porous silk groups, while only cross-linking-induced micropores
methacryloyl hydrogels were observed in non-porous counterparts. Although
The water-in-water emulsion principle, describing the NFs were detectable as nanofillers, their incorporation
phase separation that occurs when two hydrophilic did not compromise the microporous structure of SilMA
polymer solutions are mixed under specific conditions, hydrogels. In NF-containing groups, NFs were primarily
was applied in this study to create pores in the hydrogel. localized within the hydrogel matrix and on pore walls,
This phenomenon requires polymer concentrations to resulting in an increased surface roughness compared to
exceed a critical threshold (1.6% w/v for PEO was chosen NF-free samples. Notably, the 1%NF/SilMA and 2%NF/
based on previous studies), disrupting thermodynamic SilMA groups exhibited limited NF dispersion areas with
equilibrium and forming immiscible aqueous phases. visible aggregation and surface pits. With the introduction
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Volume 11 Issue 4 (2025) 283 doi: 10.36922/IJB025140118