Skeletal muscle tissue engineering (SMTE) aims at the in vitro generation of 3D skeletal muscle engineered constructs which mimic the native muscle structure and function. Although native skeletal muscle is a highly dynamic tissue, most research approaches still focus on static cell culture methods, while research on stimulation protocols indicates a positive effect, especially on myogenesis. A more mature muscle construct may be needed especially for the potential applications for regenerative medicine purposes, disease or drug disposition models. Most efforts towards dynamic cell or tissue culture methods have been geared towards mechanical or electrical stimulation or a combination of those. In the context of dynamic methods, pulsed electromagnetic field (PEMF) stimulation has been extensively used in bone tissue engineering, but the impact of PEMF on skeletal muscle development is poorly explored. Here, we evaluated the effects of PEMF stimulation on human skeletal muscle cells both in 2D and 3D experiments. First, PEMF was applied on 2D cultures of human myoblasts during differentiation. In 2D, enhanced myogenesis was observed, as evidenced by an increased myotube diameter and fusion index. Second, 2D results were translated towards 3D bioartificial muscles (BAMs). BAMs were subjected to PEMF for varying exposure times, where a 2-h daily stimulation was found to be effective in enhancing 3D myotube formation. Third, applying this protocol for the entire 16-days culture period was compared to a stimulation starting at day 8, once the myotubes were formed. The latter was found to result in significantly higher myotube diameter, fusion index, and increased myosin heavy chain 1 expression. This work shows the potential of electromagnetic stimulation for enhancing myotube formation both in 2D and 3D, warranting its further consideration in dynamic culturing techniques.
For tissue engineering of skeletal muscles, there is a need for biomaterials which do not only allow cell attachment, proliferation, and differentiation, but also support the physiological conditions of the tissue. Next to the chemical nature and structure of the biomaterial, its response to the application of biophysical stimuli, such as mechanical deformation or application of electrical pulses, can impact in vitro tissue culture. In this study, gelatin methacryloyl (GelMA) is modified with hydrophilic 2‐acryloxyethyltrimethylammonium chloride (AETA) and 3‐sulfopropyl acrylate potassium (SPA) ionic comonomers to obtain a piezoionic hydrogel. Rheology, mass swelling, gel fraction, and mechanical characteristics are determined. The piezoionic properties of the SPA and AETA‐modified GelMA are confirmed by a significant increase in ionic conductivity and an electrical response as a function of mechanical stress. Murine myoblasts display a viability of >95% after 1 week on the piezoionic hydrogels, confirming their biocompatibility. The GelMA modifications do not influence the fusion capacity of the seeded myoblasts or myotube width after myotube formation. These results describe a novel functionalization providing new possibilities to exploit piezo‐effects in the tissue engineering field.
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