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Page 4 of 16 Wang et al. Microstructures 2023;3:2023042 https://dx.doi.org/10.20517/microstructures.2023.46

Preparation and characterization of agarose/Fe O hydrogels and gelatin/Fe O scaffolds
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The agarose/Fe O hydrogels were prepared by dispersing the citrate-modified Fe O NPs in agarose
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hydrogels. At first, 0.1 g agarose was dissolved in 5 mL PBS at 110 °C. After the temperature of the agarose
solution was cooled to around 40 °C, the citrate-modified Fe O NPs suspension solution was added to
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prepare 1% agarose solution with Fe O NPs at 5 mg mL , 10 mg mL , and 20 mg mL . Agarose hydrogels
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without Fe O NPs were prepared as a control. After vortexing, the mixture was added into cylinder molds
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(Φ10 mm × H4 mm) and immediately transformed into a 4 °C refrigerator for 30 min to form agarose/Fe O
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hydrogels. The obtained hydrogels with different NP concentrations were referred to as agarose/Fe O -5
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(5 mg mL ), agarose/Fe O -10 (10 mg mL ), and agarose/Fe O -20 (20 mg mL ).
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The porous scaffolds of gelatin and Fe O NPs were prepared by using ice particulate porogen methods [51-61] .
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Firstly, ice particulates with a diameter of 250-355 μm were obtained by spraying Milli-Q water into liquid
nitrogen and sieved in a low-temperature chamber. Gelatin solution (8%, wt/v) in 70% acetic acid was
mixed with the citrate-modified Fe O NP suspension solution (1:1, v/v) to obtain gelatin/Fe O NP mixture
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solution. Then, the temperature-balanced ice particulates (-4 °C) were added to the gelatin/Fe O NP
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mixture solution (7:3, wt/v) in a -4 °C chamber, and the final concentrations of Fe O NPs in the mixture
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solution were 5, 10, and 20 mg cm , respectively. The mixture was transformed into a silicone frame and
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frozen at -20 °C for 12 h and -80 °C for 4 h. Then, the lyophilized constructs were cross-linked by
EDC (50.0 mM) and NHS (20.0 mM) in a series of ethanol/water mixture solvents [95/5, 90/10, and 85/5 (v/
v)] each for 8 h. Finally, the cross-linked scaffolds were immersed in 0.1 M glycine solution to block the
activated residual carboxyl groups. The lyophilized gelatin/Fe O NPs composite scaffolds prepared with
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different concentrations of citrate-modified Fe O NPs were referred to as gelatin/Fe O -5 (5 mg cm ),
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gelatin/Fe O -10 (10 mg cm ), and gelatin/Fe O -20 (20 mg cm ). A control scaffold without Fe O NPs was
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also prepared and referred to as a gelatin porous scaffold. An optical microscope (Olympus, Japan) and a
scanning electron microscope (SEM, Hitachi S-4800, Tokyo, Japan) were used to observe the gross
appearances and the inner pore structures of the lyophilized agarose/Fe O hydrogels and gelatin/Fe O
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scaffolds.
Magnetic thermal property of Fe O NPs in different matrices
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The magnetic thermal properties of free Fe O NPs, agarose/Fe O hydrogels, and gelatin/Fe O porous
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scaffolds were investigated under AMF irradiation. First, 300 μL of free Fe O -5, Fe O -10, and Fe O -20
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solutions were added in 0.5 mL Eppendorf tubes, respectively. The samples were placed in the center of
Double H CoilSets AMF (Frequency: 373.6 kHz; Field intensity: 130 Gauss) for 10 min using a D5 series
machine (nB nanoScale BioMagnetics, Zaragoza, Spain). Subsequently, the IR1 thermal imaging system (nB
nanoScale Biomagnetics, Zaragoza, Spain) was used to record the temperature change of different samples.
Triplicate samples were used for each measurement.
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Similarly, 300 μL of aqueous agarose solution without or with 5 mg mL , 10 mg mL , and 20 mg mL of
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Fe O NPs were added in 0.5 mL Eppendorf tubes, respectively. All samples were immediately transformed
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into a 4 °C refrigerator for 30 min to form agarose hydrogel, agarose/Fe O -5, agarose/Fe O -10, and
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agarose/Fe O -20 hydrogel samples. Before exposure to AMF, the agarose/Fe O hydrogel samples were
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balanced at room temperature for 2 h. The temperature change of the agarose/Fe O hydrogel samples was
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measured and recorded during 10 min AMF irradiation at 373.6 kHz of frequency and a field intensity of
130 Gauss. Triplicate samples were used for each measurement.
The gelatin/Fe O porous scaffolds were molded into cylinder discs (Φ10 mm × H4 mm) and hydrated with
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pure water (300 μL/disc) in silicone frames. The gelatin/Fe O porous scaffold discs were exposed to AMF
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for 10 min (Frequency: 373.6 kHz; Field intensity: 130 Gauss), and the temperature change was measured.