Fibrous scaffolds with dual porosity for the in vitro engineering of skeletal muscle tissue

In cases where muscle injuries result in damage surpassing the natural regenerative capacity of skeletal muscle tissue, a surgical approach is necessary, typically involving prosthetic implantation or the grafting of material from a donor. This approach often brings complications such as donor site morbidity, a shortage of material for autografts, or the lifelong requirement for immunosuppressant therapy to prevent rejection in the case of allografts. To tackle this issue, recent research has concentrated on the in-vitro engineering of grafts that employ patient-derived skeletal muscle cells to populate a three-dimensional structure capable of mimicking physiological tissue. These constructs could also serve to develop in-vitro skeletal muscle tissue models. This study focused on designing, developing, and characterizing a scaffold with interconnected pores to ensure uniform nutrient supply and waste removal. Aligned microfibers were incorporated to serve as topological cues for cell attachment and proliferation. The porous scaffold was designed through Fused Deposition Modelling (FDM) and porogen leaching techniques. Polymer blends of Poly-ε-caprolactone (PCL) and polyethylene Glycol (PEG) were used with different composition. The blends were prepared by solvent casting and subjected to thermal and chemical characterization through DSC and ATR-FTIR analysis before FDM processing. The scaffold design consisted of 2 layers, with a total height of 0.4 mm, and tested fill densities of 27% and 33%. Post-printing evaluation revealed acceptable shape fidelity, with only a slight increase in filament thickness compared to CAD model. Leaching was performed in demineralized water for three days, during which PEG was completely removed, as confirmed by ATR-FTIR analysis. Mechanical characterization was performed by tensile testing, with post-leaching blends showing slightly lower Young’s moduli. SEM images were taken at various magnifications for morphological evaluation of the leached scaffolds, to select one blend composition. Finally in vitro cell culture experiments were performed on scaffolds functionalized with different biomolecules, to select that functionalization leading to enhanced cell alignment, adhesion, proliferation. Future work will explore the role of mechanical and/or electrical stimulations in tissue maturation.