Micro/nano-hierarchical scaffold fabricated using a cell electrospinning/3D printing process for co-culturing myoblasts and HUVECs to induce myoblast alignment and differentiation

Human skeletal muscle is composed of intricate anatomical structures, including uniaxially arranged myotubes and widely distributed blood capillaries. In this regard, vascularization is an essential part of the successful development of an engineered skeletal muscle tissue to restore its function and physiological activities. In this paper, we propose a method to obtain a platform for co-culturing human umbilical vein endothelial cells (HUVECs) and C2C12 cells using cell electrospinning and 3D bioprinting. To elaborate, on the surface of mechanical supporters (polycaprolactone and collagen struts) with a topographical cue, HUVECs-laden alginate bioink was uniaxially electrospun. The electrospun HUVECs showed high cell viability (90%), homogeneous cell distribution, and efficient HUVEC growth. Furthermore, the myoblasts (C2C12 cells), which were seeded on the vascularized structure (HUVECs-laden fibers), were co-cultured to facilitate myoblast regeneration. As a result, the scaffold that included myoblasts and HUVECs represented a high degree of the myosin heavy chain (MHC) with striated patterns and enhanced myogenic-specific gene expressions (MyoD, troponin T, MHC and myogenin) as compared to the scaffold that included only myoblasts. Cell electrospinning is an advanced electrospinning method that improves cell–matrix interactions by embedding cells directly into micro/nanofibers. Here, cell electrospinning was employed to achieve not only the homogeneous human umbilical vein endothelial cells (HUVECs) distribution with a high cell-viability (~90%), but also highly aligned topographical cue. Moreover, the uniaxially micropatterned PCL/collagen struts as a physical support were generated using three-dimensional (3D) printing, and was covered with HUVEC-laden micro/nanofibers. This hierarchical structure provided meaningful mechanical stability, homogeneous cell distribution, and HUVEC transformation into a narrow, elongated structure. Furthermore, the myoblasts (C2C12 cells) were seeded on the HUVECs-laden fibers and cocultured to facilitate myogenesis. In brief, a myosin heavy chain with striated patterns and enhanced myogenic specific gene expressions were represented.