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Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and β-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After three days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion as well as S6 activation were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic or regenerating skeletal muscle.

Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates (“2D”) or embedded in fibrin hydrogels (“3D”) with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels’ fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7–30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.

Classical approaches to engineer skeletal muscle tissue based on current regenerativeand surgical procedures still do not meet the desired outcome for patient applications.Besides the evident need to create functional skeletal muscle tissue for the repairof volumetric muscle defects, there is also growing demand for platforms to studymuscle-related diseases, such as muscular dystrophies or sarcopenia. Currently,numerous studies exist that have employed a variety of biomaterials, cell types andstrategies for maturation of skeletal muscle tissue in 2D and 3D environments. However,researchers are just at the beginning of understanding the impact of different culturesettings and their biochemical (growth factors and chemical changes) and biophysicalcues (mechanical properties) on myogenesis. With this review we intend to emphasizethe need for new in vitro skeletal muscle (disease) models to better recapitulate importantstructural and functional aspects of muscle development. We highlight the importance ofchoosing appropriate system components, e.g., cell and biomaterial type, structural andmechanical matrix properties or culture format, and how understanding their interplay willenable researchers to create optimized platforms to investigate myogenesis in healthyand diseased tissue. Thus, we aim to deliver guidelines for experimental designs to allow estimation of the potential influence of the selected skeletal muscle tissue engineeringsetup on the myogenic outcome prior to their implementation. Moreover, we offer aworkflow to facilitate identifying and selecting different analytical tools to demonstratethe successful creation of functional skeletal muscle tissue. Ultimately, a refinement ofexisting strategies will lead to further progression in understanding important aspectsof muscle diseases, muscle aging and muscle regeneration to improve quality of life ofpatients and enable the establishment of new treatment options.&nbsp

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca2+ dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Statement of Significance Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.