A recent article published in Advanced Materials Technologies demonstrated the generation of complex skeletal muscle-like tissues using three-dimensional (3D) bioprinting of a Gelatin-methacrylate (GelMA)-based ink on a polymeric magnetic actuator.
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Background
Skeletal muscle tissue can self-repair minor injuries, but significant muscle loss leads to non-functional scar tissue. Current treatments mainly involve autologous muscle transfer. Tissue engineering research offers an alternative, allowing the creation and maturation of cell-laden biocompatible scaffolds in the laboratory.
3D bioprinting of complex structures has gained attention in several fields, including dentistry, pharmaceuticals, medical devices, and tissue/organ engineering. However, it faces challenges such as decreased cell viability, prolonged printing time, and difficult print shape preservation.
Additionally, formulating bio-inks to generate complex engineered structures with desired geometry and cell viability is challenging.
To address these issues, researchers designed magnetically responsive platforms to induce non-invasive shape transformation after 3D printing in C2C12 myoblast-laden bio-ink comprising gelatin and alginate.
Methods
Type A pigskin gelatin was used to prepare GelMA through the dialysis process. The prepared GelMA was photo-cross-linked with alginate in different compositions to optimize printability, cross-linking efficiency, and cell viability.
Magnetically responsive platforms were fabricated using two different 3D-printed molds, with and without a folding neck. The molds were supplemented with carbonyl magnetic Fe particles.
After curing, the platforms were peeled off the molds and modified with (3-aminopropyl)triethoxysilane molecules to overcome their hydrophobicity. All magnetic actuation experiments on these platforms were performed with permanent magnets of varying strengths.
Four different bio-inks were used to 3D print the cell-laden scaffolds in three distinct patterns. A permanent magnet was rolled on the platforms with the cell-laden scaffold, which was then cultured under standard cell culture conditions using a high-glucose medium.
Alginate was selectively removed from the scaffold using alginate lyase (AL) enzyme to promote cell spreading within the matrix. The wells were then supplemented with a low-glucose differentiation medium and cultured for seven days.
Tensile tests were conducted to evaluate the modulus of elasticity of the three cell-laden 3D-printed scaffolds (length: 14 mm, cross-sectional area: 3 mm2) on the seventh day, both with and without AL treatment. Scanning electron microscopy was employed to examine the morphology and porosity of the scaffolds before and after AL treatment.
Cell viability was assessed with the AlamarBlue cell proliferation assay. Additionally, cell morphology in the scaffolds was examined using a fluorescence microscope.
Myogenic differentiation of C2C12s was investigated by immunofluorescent staining for MyoD1. All quantitative results were presented as mean ± standard deviation (n = 3).
Results and Discussion
The 3D-printed magnetic EcoFlex platforms with cell-laden scaffolds exhibited shape-morphing capability, highlighting the importance of proper design considerations to achieve reproducible and controlled outcomes in such systems.
Tensile test results indicated that removing alginate from the system adjusted the scaffold's stiffness to a suitable range for C2C12 cells. Fluorescent microscopy on the first day observed normal cell and nucleus morphology. However, by the seventh day, images revealed compromised mechanical integrity, indicating cell proliferation and irregular spread within some scaffolds.
Immunofluorescence analysis revealed that C2C12 cells formed myotubes after seven days, indicating successful muscle tissue differentiation. The C2C12 cells remained viable upon controlled shape transformation, with functional myotube formation initiated within bio-printed platforms. Notably, rolled scaffolds exhibited enhanced MyoD1 expression compared to open scaffolds.
The geometric shape of the designed patterns aligned parallel to the direction of cell spreading, indicating significant potential to control cell alignment by modifying scaffold pattern dimensions and shapes. More myotube-like structures were observed on patterns with a lower initial cell population, suggesting that spatial confinement and cell-cell interactions within a limited region significantly promote myotube formation.
Conclusion
This study successfully demonstrated the fabrication and utilization of GelMA as a photo-cross-linkable material combined with alginate to create a bio-ink suitable for 3D printing. The prepared biomaterial served as a cell carrier scaffold, providing a conducive environment for cell adhesion, proliferation, and differentiation.
Despite challenges in scaffold fabrication and magnetic folding, the cells maintained viability, proliferating in the GelMA-alginate structure for seven days. The dynamic behavior of the GelMA scaffold allowed for the alignment and organization of cells within the rolled structure.
The results validate the potential of the proposed methodology in generating complex structures using a simple magnetic actuation procedure. The researchers suggest that this magnetic actuation can be used to design repeatable self-switching systems, operating non-invasively to alter shapes.
Fabricating highly sophisticated and functional tissue constructs using this methodology can ultimately advance regenerative medicine.
Journal Reference
Ergene, E., Liman, G., Yilgor, P., Demirel, G. (2024). Magnetically Actuated GelMA‐Based Scaffolds as a Strategy to Generate Complex Bioprinted Tissues. Advanced Materials Technologies. doi.org/10.1002/admt.202400119
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