Elucidating molecular events underlying topography mediated cardiomyogenesis of stem cells on 3D nanofibrous scaffolds.

Toward engineering a cardiac patch, the objective of this work was to assess stem cell response to a three-dimensional (3D) nanofibrous scaffold and probe the underlying molecular mechanisms including both cell signaling and epigenetic changes. Cardiomyogenesis of human mesenchymal stem cells (hMSCs) in 3D poly(ε-caprolactone) (PCL) nanofibers and macroporous scaffolds was compared with two-dimensional (2D) PCL films. In addition, nanofiber mats of PCL and its blend with gelatin (PCL-Gel) were prepared with fibers of random or unidirectional alignment to assess the roles of topography (fibrous architecture and its alignment) and biochemical cue (cell-adhesive sites) in directing cell functions. Cells on 3D random nanofibers, exhibited elevated expression of known cardiac markers such as cardiac actinin, cardiac troponin and β-myocardial heavy chain compared to cells on 2D films suggesting enhanced differentiation that was further accentuated on the aligned fibers. 3D macroporous scaffolds did not enhance the cardiomyogenic differentiation. However, minimal differences were noted between cells on PCL and PCL-Gel fibers, irrespective of alignment. Co-culture with neonatal rat cardiomyocytes induced beating in the differentiated cells. The use of small molecule inhibitors revealed that cytoskeletal elements F-actin, microtubules and downstream ROCK protein are essential for the cardiomyogenesis of hMSCs on the nanofibers. The activation of ERK, AKT and mTOR was observed during cardiomyogenesis. Interestingly, enhanced differentiation on the aligned nanofibers was associated with increased level of the histone deacytelase SIRT6 and decreased level of the acetylated histone H3K9 suggesting a role for epigenetic regulation. This study demonstrates that aligned nanofibrous scaffolds augment cardiomyogenic differentiation wherein topography plays a critical role in driving stem cell function. In addition, this study offers insight into molecular pathways driving the cellular response.