Gelatin methacryloyl hydrogel with post-injection magnetic alignment: Structural modulation and protein-cell...

Gelatin methacryloyl (GelMA), a photocrosslinkable derivative of collagen-derived gelatin, retains inherent bioactive motifs including RGD sequences for cell adhesion and matrix metalloproteinase-sensitive domains for enzymatic remodeling. However, conventional GelMA hydrogels exhibit isotropic architectures that fail to recapitulate the anisotropic organization of native tissues. Here, we report a strategy to introduce controlled fibril alignment and structural organization into injectable GelMA hydrogels through post-injection magnetic alignment of embedded polycaprolactone (PCL) microfibrils containing magnetic nanoparticles (MNPs). This approach enables fibril orientation within 30 s under external magnetic field, followed by UV crosslinking to preserve the aligned architecture without compromising the protein-based matrix integrity. Aligned architectures exhibited enhanced compressive modulus (∼11 kPa at 0.1% fibril content) comparable to random configurations with tenfold higher loadings, demonstrating that structural organization outweighs material quantity in determining bulk stiffness. In C2C12 myoblast cultures within the GelMA matrix, fibril alignment promoted cell elongation and moderate directional organization, though responses remained heterogeneous. Interestingly, while mechanical reinforcement was evident at the lowest fibril content, significant upregulation of MyoD occurred only at ≥ 0.5%, suggesting that cellular responses to the protein-based matrix depend on cell-fibril contact frequency rather than bulk mechanics alone. These findings suggest that structural modulation of gelatin-based biopolymer hydrogels can influence bulk compressive stiffness and early protein-cell interactions, providing insights into structure-function relationships in aligned biopolymer systems.