The essence of biophysical cues in skeletal muscle tissue engineering

Skeletal muscle is an appealing topic for tissue engineering because of its variety in applications. Evidently, tissue engineered skeletal muscle can be used in the field of regenerative medicine to repair muscular defects or dystrophies. Engineered skeletal muscle constructs can also be used as a model system for drug-screening or to study muscle physiology and the etiology of pressure sores. Besides these well known applications, a new field of interest with high societal impact has arisen, being in vitro cultured meat. Contemporary large-scale farming and transportation of livestock brings along a high risk of infectious animal diseases and environmental burden through greenhouse gas emission. In vitro cultured meat can dramatically reduce these risks and improve animal welfare. Although major advances have been made so far in skeletal muscle tissue engineering, the maturation level of the engineered muscle constructs is still not satisfactory. The requirements of the engineered skeletal muscle constructs are similar for both regenerative medicine and in vitro meat production: mature functional skeletal muscle tissue is required that can produce force within the physiological range. To improve the maturation process of skeletal muscle progenitor cells we have focused on mimicking the native environment of these cells. In particular, we have focused on biophysical cues that play an essential role in the regeneration of skeletal muscle tissue in vivo. These cues were investigated in conventional 2D cultures, as well as in 3D model systems that are physiologically more relevant. An important biophysical cue that was investigated is electrical stimulation, since nerve stimulation is known to be a prerequisite for myotube formation in vivo. We have shown that electrical stimulation results in an acceleration of the formation of cross striations and increased expression levels of muscle maturation markers in 2D and 3D experiments. These effects were observed in cultures of the conventional C2C12 cell line and primary muscle progenitor cells (MPCs). More specifically, electrical stimulation of 3D cultures with MPCs resulted in a shift of myosin heavy chain (MHC) expression towards slower isoforms. Electrical stimulation can be implemented in skeletal muscle tissue engineering strategies to improve efficiency of the culture process and to tune MHC expression, which may be relevant for the final texture of the engineered constructs. Mechanical cues also play an important role in muscle development in vivo, both in the embryonic phase and in adults. Stretch can result in hypertrophy of skeletal muscle tissue and can therefore improve texture and force production of tissue engineered skeletal muscle constructs. However, our mechanical stimulation protocol, with applied strains within the physiological range, resulted in impaired muscle maturation in both C2C12 and primary MPCs and is therefore not useful for skeletal muscle tissue engineering. A major finding of the results presented in this thesis is that the 3D environment in which muscle progenitor cells are cultured is essential for myogenesis. Sarcomere formation was faster in a 3D hydrogel based environment, compared to conventional 2D cultures. In 2D, sarcomere formation is optimal on substrates with a stiffness similar to in vivo skeletal muscle tissue, between 3-12 kPa. However, the stiffness of our 3D hydrogel systems was considerably lower than this range and muscle formation still progressed rapidly. We concluded that not the substrate stiffness itself, but the ability of cells to develop tension is essential for the formation of cross striations. Both in 2D and 3D settings we demonstrated that the Rho-associated kinase plays a role in this process, since no cross striations were observed when this kinase was inhibited. Additionally, 3D culture methods that enable an increase in cellular tension result in acceleration of the maturation process. MPCs cultured in 3D resulted in more mature skeletal muscle tissue compared to C2C12 and are therefore the preferred cell source for tissue engineering applications. However, their proliferative capacity remains limited. We showed that the C2C12 cell line, which is readily accessible and easy to culture and differentiate, can be used as a model system to design 3D culture methods and biophysical stimulation regimes. In conclusion, we have shown that several biophysical cues are important for in vitro skeletal muscle maturation. The results presented in this thesis have contributed to the technology that can realize the in vitro production of meat.