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Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe3O4), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk‐fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X‐ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real‐time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION‐casein scaffolds including GATA‐4, cardiac troponin T, Nkx 2.5, and alpha‐myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core‐shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self‐renewal capacity.

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