Application Cases

Experiment Name: Preparation of Multifunctional Gelatin Composite Hydrogel and In Vitro Construction of Engineered Cardiac Tissue

Research Direction: Biomedical Engineering

Test Objective:
To prepare a three-dimensional anisotropic electromagnetic functional composite hydrogel scaffold based on GelMA (GelMA-PDA-rGO-Fe3O4). This scaffold is fabricated by directionally solidifying  PDA-rGO-Fe3O4 electromagnetic bifunctional nanocomposites (Fe3O4 nanoparticles modified PDA-rGO) under a DC magnetic field. 2D and 3D cell culture results demonstrated that the unique anisotropic structure facilitates the directed growth  and beating of cardiomyocytes on the surface. Due to the composite hydrogel's combined electrical conductivity and magnetic properties, magnetic stimulation studies using both DC and AC magnetic fields were conducted. The results indicated that AC magnetic field stimulation, similar to electrical stimulation, promotes the development of a  complete  cytoskeletal structure, increases the beating rate and frequency of cardiomyocytes, and upregulates the expression of cardiac-specific proteins and maturation-related genes. The engineered cardiac tissue (ECT) constructed based on GelMA-PDA-rGO-Fe3O4 exhibited butterfly-like fluttering beating behavior during long-term culture, potentially serving as a biological actuator for visualized drug screening. In summary, the multifunctional PDA-rGO-Fe3O4 composite hydrogel proves to be an excellent scaffold material with potential applications in cardiac repair, drug screening, and disease modeling.

Testing Equipment: ATA-304 Power Amplifier, Signal Generator, Helmholtz Coil, Cell-Seeded Well Plates, Hydrogel Scaffold.

Experimental Procedure:
Prior to the experiment, the electromagnetic functional PDA-rGO-Fe3O4 nanocomposite and the anisotropic GelMA-PDA-rGO-Fe3O4 electromagnetic composite hydrogel were prepared. Subsequently, cardiomyocytes were extracted, seeded (2D and 3D), and cultured. Finally, the cardiomyocytes were subjected to electrical and magnetic stimulation treatments.

Figure 4.1: Physical Diagram of the Static Magnetic Field Generation Device

1. DC Magnetic Field Stimulation
DC magnetic field stimulation of cardiomyocytes was performed using a DC magnetic field generation device, schematically shown in Figure 4.1. The cell-seeded well plate was placed within a uniform magnetic field region. When the DC current was 0.87 A, the generated DC magnetic field  intensity  was 1.47 mT, consistent with the theoretical value of 1.5 mT.

2. AC Magnetic Field Stimulation
As the name implies, AC magnetic field stimulation of cardiomyocytes was performed using an AC magnetic field generation device, schematically shown in Figure 4.2. Unlike the DC device, the AC magnetic field was generated by driving the coil with an amplified sinusoidal current output jointly by a signal generator and a power amplifier. When the applied sinusoidal wave frequency was 5 Hz and the current was 1 A, the magnetic field  intensity  was approximately 1 mT. Cardiomyocytes were statically cultured for 3 days, then placed in a uniform AC magnetic field for continuous stimulation for 5 days. After removing the magnetic field, static culture was continued for another 7 days. An experimental group without magnetic field stimulation served as a positive control, and GelMA-Fe3O4 served as a negative control.

Figure 4.2: Schematic Diagram of the AC Magnetic Field Generation Device

3. Effect of Applying AC Magnetic Stimulation on Cardiomyocyte Function
The GelMA-PDA-rGO-Fe3O4 magnetic composite hydrogel was used in conjunction with external magnetic stimulation to construct mature ECT for future research in cardiac patches and drug screening.

Figure 4.17: AC Magnetic Field Stimulation Device for Cardiomyocyte Magnetic Stimulation. (A) Magnetic stimulation protocol for promoting cardiomyocyte maturation. (B) Schematic diagram of the magnetic stimulation device, including Helmholtz coils, a signal generator, and a power amplifier. After magnetic stimulation and culture for 15 days, (C) cell contraction vector maps of cardiomyocytes in each group, with vectors represented in red between two frames, and (D) corresponding beating signal recording curves. Scale bar: 500 μm. Corresponding statistical evaluation of (E) spontaneous beating frequency and (F) beating rate for each group. *p<0.05 and **p<0.01.

As shown in Figure 4.17A, neonatal rat cardiomyocytes were seeded onto the hydrogel scaffolds, statically cultured for 3 days, then placed in an AC magnetic field device (magnetic induction intensity of 1 T, Figure 4.17B) for continuous stimulation for 5 days. The magnetic field was removed on day 8, followed by another week of static culture, totaling 15 days. Figures 4.17C and 4.17D illustrate the beating behavior of cardiomyocytes in the stimulated and control groups on day 15 of culture. Due to the symmetric folding tendency of the anisotropic GelMA-PDA-rGO-Fe3O4 composite hydrogel scaffold, the cardiomyocytes on its surface contracted synchronously in the same direction. In contrast, cardiomyocytes on the GelMA group and isotropic composite group scaffolds beat synchronously in random directions due to the asymmetric folding of the scaffold material. Furthermore, the average beating frequency (BPM) in the stimulated composite groups (both anisotropic and isotropic) on day 15 remained higher than that in the GelMA stimulated group, similar to the observations on day 8 of static culture. As shown in Figure 4.17E, the average BPM for each group was 44±2 (GelMA group), 60±3 (isotropic composite group), and 76±4 (anisotropic composite group), essentially consistent  with the average BPM values on day 8, indicating that magnetic stimulation has a relatively minor effect on the beating frequency of cardiomyocytes.

Experimental Results:
Following magnetic stimulation and long-term culture, the beating rate of cardiomyocytes in all groups increased significantly, reaching mm/s, making the spontaneous beating of the generated cardiac tissue visible to the naked eye. As shown in Figures 4.17D and 4.17F, the quantitative contraction rates were 4.07±0.18 mm/s (GelMA group), 9.48±0.46 mm/s (isotropic composite group), and 12.40±0.63 mm/s (anisotropic composite group), and the relaxation rates were 2.36±0.11 mm/s (GelMA group), 3.27±0.16 mm/s (isotropic composite group), and 11.97±0.62 mm/s (anisotropic composite group). Therefore, our results demonstrate that the synergistic effect of the 3D anisotropic scaffold structure induced by the PDA-rGO-Fe3O4 nanocomposite and the magnetic stimulation promotes rapid action potential propagation and electrical coupling between cardiomyocytes, driving the formation of mature ECT.

Figure: ATA-304C Power Amplifier Specifications and Parameters