Aligned nanofiber scaffolds combined with cyclic stretch facilitate mesenchymal stem cell differentiation for ligament engineering

Figure 3 Bone marrow-derived mesenchymal stem cell seeding, morphology, and viability on aligned and random nanofiber scaffolds. A: Schematic illustration of bone marrow-derived mesenchymal stem cell (BMSC) seeding on aligned and random nanofiber scaffolds placed in culture wells; B: Image of culture wells containing scaffolds with uniform BMSC seeding; C: Scanning electron microscopy images of aligned (top) and random (bottom) nanofiber scaffolds, highlighting organized orientation in aligned fibers and disordered structure in random fibers. Scale bars: 50 μm and 20 μm; D: Immunofluorescent staining for F-actin (red) and nuclei (4’,6-diamidino-2-phenylindole, blue) illustrating BMSC morphology and cytoskeletal arrangement on aligned vs random scaffolds. BMSCs on aligned scaffolds show an elongated, aligned cytoskeletal organization, while those on random scaffolds exhibit a more dispersed morphology; E: Cell viability assay using CCK-8 over 7 days, showing OD values at 450 nm for BMSCs on aligned, random, and control surfaces; F: Doubling time analysis of BMSCs on each substrate, with aligned scaffolds supporting faster proliferation than the random and control conditions. ^bP < 0.01, NS: Not significant. DAPI: 4’,6-diamidino-2-phenylindole.