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

Figure 5 Effects of Ras homolog gene family (Rho)-associated coiled coil-containing kinase inhibition with Y27632 on bone marrow-derived mesenchymal stem cell tenogenic differentiation and cytoskeletal organization under static and tensile conditions. A: Schematic showing the experimental setup for culturing bone marrow-derived mesenchymal stem cell (BMSCs) on aligned and random nanofiber scaffolds under static and tensile conditions with Y27632 treatment; B: Immunofluorescent staining of F-actin (red) and nuclei (4’,6-diamidino-2-phenylindole, blue) illustrating the cytoskeletal alignment of BMSCs in response to static and tensile conditions with Y27632. Under tensile strain, BMSCs on both scaffold types align along the strain direction (yellow arrows). Scale bars: 50 μm; C: Western blot analysis of tenogenic proteins (collagen I, collagen III, tenascin C, and tenomodulin) in BMSCs on aligned and random scaffolds under static and tensile conditions with Y27632, with β-actin as the loading control; D: Quantification of protein expression levels, showing scaffold orientation and mechanical strain effects on tenogenic protein expression in the presence of Y27632; E: Expression analysis of Ras homolog gene family (Rho)-associated coiled coil-containing kinase, focal adhesion kinase, and runt-related transcription factor 2 at both gene and protein levels, demonstrating the effects of Y27632 on tenogenic signaling pathways in BMSCs under static and tensile conditions on aligned and random scaffolds. ^aP < 0.05, ^bP < 0.01. DAPI: 4’,6-diamidino-2-phenylindole; ROCK: Ras homolog gene family (Rho)-associated coiled coil-containing kinase; FAK: Focal adhesion kinase; RUNX2: Runt-related transcription factor 2.