Myogenic Differentiation on 3D‐Printed Interpenetrating Bioactive Hydrogel of Decellularised Human Placenta With Fish Scale Gelatin

ABSTRACT Tissue‐specific templates are essential for muscle differentiation as they enhance cell alignment and regeneration. Advancements in extrusion 3D printing with biomaterial inks enable customizable designs and tailored spatial arrangements. Key growth factors and extracellular matrix (ECM) constituents are crucial for muscle differentiation from stem cells, with the placenta being a significant resource. This study explores human placenta ECM‐fish gelatin‐based hydrogels as a cost‐effective alternative. ECM was obtained via ultrasonication‐assisted decellularisation, which preserved its structure while removing cellular content. The research focused on placenta‐gelatin ink formulations for scaffold printing, forming an interpenetrating network hydrogel. Rheological analysis indicated that placenta‐fish gelatin biomaterial ink exhibited a higher storage modulus compared to only fish gelatin formulations. Analytical techniques such as FESEM, FTIR, and the Ninhydrin assay confirmed that the placenta hydrogels exhibit shape fidelity and achieved approximately 75 ± 7% crosslinking density. Hydrogels supported cellular proliferation and aligned growth. Myogenic differentiation involving C2C12 cells and human amniotic membrane stem cells (HAMSCs) demonstrated organized myoblasts and aligned myotubes, respectively. CAM assay revealed enhanced angiogenesis and microvascular growth with negligible hemolysis. This research offers biowaste‐derived ECM hydrogels for skeletal muscle engineering, eliminating the need for sacrificial templates or synthetic crosslinkers.