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

the incorporated nanofibrous architectures substantially electrospinning, the NF membrane was placed in a fume
enhanced the proliferation and osteogenic differentiation of hood overnight, followed by cross-linking with absolute
bone marrow-derived mesenchymal stem cells, suggesting ethanol for 15 min and drying at room temperature.
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great potential for bone tissue engineering applications. 25 The electrospun NFs were subsequently weighed and
homogenized in deionized water to obtain a suspension of
Therefore, in this study, we developed a 3D printable,
NF-reinforced porous SilMA hydrogel designed for short NFs, which was stored at 4°C for further use.
cartilage regeneration. PEO was used as a template to 2.3. Synthesis of silk methacryloyl
increase internal pore structure, and short electrospun Briefly, degummed silk was dissolved in LiBr and reacted at
SF NFs were incorporated to enhance its mechanical and 60°C for an hour under constant stirring at 300 rpm. GMA
biological properties. A notable advantage of the water-in- (424 mM) was then added to the solution, and the mixture
water emulsion system is its inherently low and unstable was stirred for an additional 3 h for the functionalization
interfacial tension. The addition of short NFs is expected reaction between SF and GMA. The reaction mixture was
to improve emulsion stability by reducing the interfacial transferred to a dialysis membrane (molecular weight cut-
free energy. Our approach includes characterization off: 12,000–14,000) and dialyzed against deionized water
26
of the hydrogel’s physicochemical properties, in vitro for 4 days. Finally, the dialyzed solution was freeze-dried
evaluation of biocompatibility and bioactivity, and in vivo to obtain SilMA for future use. 20
assessment of chondrogenic ability through subcutaneous
implantation in non-obese diabetic/severe combined 2.4. Preparation of silk methacryloyl hydrogels
immunodeficiency mice. with poly(ethylene oxide)-induced porosity and
nanofiber reinforcement
2. Materials and methods The prepared silk NF membrane samples were homogenized
to form short NF suspensions. SilMA powder, LAP powder
2.1. Materials (Aladdin, China), and the NF suspensions were thoroughly
Silkworm cocoons were obtained from the Jiangnan mixed and dissolved in the culture medium to prepare a final
Mulberry Silkworm Base (China). Lithium phenyl solution with concentrations of 30% w/v for SilMA, 0.25%
(2,4,6-trimethylbenzoyl) phosphate (LAP), lithium w/v for LAP, and 1 and 2% w/v for NFs. This composite
bromide (LiBr), and hexafluoroisopropanol were obtained solution was used as the non-porous hydrogel precursor
from the Shanghai Aladdin Biochemical Technology solution. PEO (molecular weight = 300,000) powder was
Co., Ltd. (China), rhodamine-B, PEO, 424 mM glycidyl dissolved separately in the culture medium to prepare
methacrylate (GMA), and dimethylmethylene blue from a 1.6% w/v solution. The hydrogel precursor and PEO
Sigma–Aldrich Corporation (United States), Hoechst solutions were then mixed at a volume ratio of 2:1 to form
33258 and type II collagenase from MedChemExpress a two-phase aqueous emulsion, which served as the porous
LLC (United States), and dialysis bags from Biomedical hydrogel precursor solution. The porous hydrogel was
Instruments Inc (China). Phosphate buffered saline (PBS) prepared using UV light-induced cross-linking, followed
was purchased from Thermo Fisher Scientific Inc. (United by PEO precipitation through immersion. Regarding 3D
States), F-12 medium and fetal bovine serum from Gibco bioprinting, the hydrogel structures were fabricated using
(United States), and Live/dead cell staining kit from a digital light processing (DLP) 3D bioprinter (ZJ-BP01,
Shanghai Beyotime Bio-Tech Co., Ltd. (China). Zhongjian 3D Technology Co., China) equipped with

2.2. Extraction of silk fibroin and preparation of a 405 nm UV light source (intensity: 20 mW/cm²). The
nanofibrous film printer was integrated with a temperature-controlled vat
The silkworm cocoons were first degummed. Then, 10 g (maintained at 4°C) to prevent premature gelation of the
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of the degummed cocoons were dissolved in a 9.3 mol/L photopolymerizable hydrogel precursor. The detailed 3D
LiBr aqueous solution at 60°C with constant stirring at printing parameters are shown in Table 1.
300 rpm for 4 h. The resulting solution was dialyzed using 2.5. Characterization
a 12–14 kDa dialysis membrane for 4 days to obtain SF.
The SF was then dissolved in hexafluoroisopropanol to 2.5.1. Proton nuclear magnetic resonance and Fourier
prepare a 7% (w/w) solution, which was stirred overnight. transform infrared spectroscopy
The solution was then loaded into a 10 mL syringe with SF and SilMA were weighed and dissolved in 0.5 mL of
an 8-gauge metal needle and connected to electrospinning deuterated dimethyl sulfoxide. The resulting solutions
equipment with the following parameters: a high voltage were transferred to a nuclear magnetic resonance (NMR)
of 16 kV, a solution flow rate of 15 μL/min, and a distance tube to determine proton NMR (1H-NMR). For Fourier
of 15 cm between the needle and the collector plate. After Transform infrared spectroscopy (FTIR), SF, SilMA,

Volume 11 Issue 4 (2025) 280 doi: 10.36922/IJB025140118