No abstract

Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method.

Tissue engineered skeletal muscle has potential for application as a graft source for repairing soft tissue injuries, a model for testing pharmaceuticals, and a biomechanical actuator system for soft robots. However, engineered muscle to date has not produced forces comparable to native muscle, limiting its potential for repair and for use as an in vitro model for pharmaceutical testing. In this study, we examined the trophic effects of dexamethasone (DEX), a glucocorticoid that stimulates myoblast differentiation and fusion into myotubes, on our tissue engineered three-dimensional skeletal muscle units (SMUs). Using our established SMU fabrication protocol, muscle isolates were cultured with three experimental DEX concentrations (5, 10, and 25 nM) and compared to untreated controls. Following seeding onto a laminin-coated Sylgard substrate, the administration of DEX was initiated on day 0 or day 6 in growth medium or on day 9 after the switch to differentiation medium and was sustained until the completion of SMU fabrication. During this process, total cell proliferation was measured with a BrdU assay, and myogenesis and structural advancement of muscle cells were observed through immunostaining for MyoD, myogenin, desmin, and a-actinin. After SMU formation, isometric tetanic force production was measured to quantify function. The histological and functional assessment of the SMU showed that the administration of 10 nM DEX beginning on either day 0 or day 6 yielded optimal SMUs. These optimized SMUs exhibited formation of advanced sarcomeric structure and significant increases in myotube diameter and myotube fusion index, compared with untreated controls. Additionally, the optimized SMUs matured functionally, as indicated by a fivefold rise in force production. In conclusion, we have demonstrated that the addition of DEX to our process of engineering skeletal muscle tissue improves myogenesis, advances muscle structure, and increases force production in the resulting SMUs.

Tissue-engineered skeletal muscle holds promise as a source of graft tissue for repair of volumetric muscle loss and as a model system for pharmaceutical testing. To reach this potential, engineered tissues must advance past the neonatal phenotype that characterizes the current state of the art. In this review, we describe native skeletal muscle development and identify important growth factors controlling this process. By comparing in vivo myogenesis to in vitro satellite cell cultures and tissue engineering approaches, several key similarities and differences that may potentially advance tissue-engineered skeletal muscle were identified. In particular, hepatocyte and fibroblast growth factors used to accelerate satellite cell activation and proliferation, followed by addition of insulin-like growth factor as a potent inducer of differentiation, are proven methods for increased myogenesis in engineered muscle. Additionally, we review our recent novel application of dexamethasone (DEX), a glucocorticoid that stimulates myoblast differentiation, in skeletal muscle tissue engineering. Using our established skeletal muscle unit (SMU) fabrication protocol, timing- and dose-dependent effects of DEX were measured. The supplemented SMUs demonstrated advanced sarcomeric structure and significantly increased myotube diameter and myotube fusion compared to untreated controls. Most significantly, these SMUs exhibited a fivefold rise in force production. Thus, we concluded that DEX may serve to improve myogenesis, advance muscle structure, and increase force production in engineered skeletal muscle.

Our lab showed that administration of dexamethasone (DEX) stimulated myogenesis and resulted in advanced structure in our engineered skeletal muscle units (SMU). While administration of 25 nM DEX resulted in the most advanced structure, 10 nM dosing resulted in the greatest force production. We hypothesized that administration of 25 nM DEX during the entire fabrication process was toxic to the cells and that administration of DEX at precise time points during myogenesis would result in SMU with a more advanced structure and function. Thus, we fabricated SMU with 25 nM DEX administered at early proliferation (days 0–4), late proliferation (days 3–5), and early differentiation (days 5–7) stages of myogenesis and compared them to SMU treated with 10 nM DEX (days 0–16). Cell proliferation was measured with a BrdU assay (day 4) and myogenesis was examined by immunostaining for MyoD (day 4), myogenin (day 7), and α-actinin (day 11). Following SMU formation, isometric tetanic force production was measured. An analysis of cell proliferation indicated that 25 nM DEX administered at early proliferation (days 0–4) provided 21.5% greater myogenic proliferation than 10 nM DEX (days 0–4). In addition, 25 nM DEX administered at early differentiation (days 5–7) showed the highest density of myogenin-positive cells, demonstrating the greatest improvement in differentiation of myoblasts. However, the most advanced sarcomeric structure and the highest force production were exhibited with sustained administration of 10 nM DEX (days 0–16). In conclusion, alteration of the timing of 25 nM DEX administration did not enhance the structure or function of our SMU. SMU were optimally fabricated with sustained administration of 10 nM DEX.