There are several attempts to induce fusion of myoblasts to myotubes in vitro, imitating the in vivo conditions during myogenesis. Critical issues include an understanding of the effects of mechanical and electrical stimulation on cultured myoblasts and the role of the extracellular matrix (ECM) in the migration, proliferation and differentiation of the cells [5, 21, 24]. Mechanical stimulation is one important factor during myogenesis which influences gene regulation, endogeneous protein expression, protein acculmulation and metabolic activity. [35-37]. Both passive and active mechanical forces play an important role in the transition of skeletal muscle from the embryonic to the mature state .
Directed mechanical tension is important to organize myoblasts into functional aligned myotubes and provides a stimulus for the expression of mature isoforms of myofibrillar proteins . Besides it has been shown that mechanical forces also have important impact in mature skeletal muscle on myofiber diameter, cell number and myofiber composition [36, 39]. Based on this knowledge Powell et al reported the development of three dimensional human skeletal muscle tissue using a 3D-scaffold based on collagen and Matrigel® by mechanical stimulation . This model allows to determine the cellular effects of mechanical stimulation, particulary those associated with cytoskeletal rearrangements. In order to improve the ratio of muscle fibers and extracelluar matrix Powell and coworkers created a mechanical cell stimulator that is able to stretch and relax the cell cultures in vitro. A force transducer was able to measure passive forces and viscoelastic properties. The mechanical stimulation improved the structure of the engineered skeletal muscle by increasing the mean myofiber diameter and the elasticity. However, the tissue that resulted on these studies is still not an appropriate substitute for functional implantation in vivo. Other studies focussing on the in vitro creation of skeletal muscle showed also a quite different morphologic and functional appearance without mechanical stimulation in comparison to native skeletal muscle. The extracellular matrix content was significantly higher, myofiber density was low and maturation was incomplete without stimulation [21, 28]. To summarize, the cellular effects of applied mechanical force seem to be an important aspect to the in vitro development of differentiated functional muscle tissue. Another approach of developing a higher differentiated and more functional skeletal muscle tissue is the application of electrical stimulation [28, 40–42]. This mimics the nerve stimulation during myogenensis and during regeneration of injured skeletal muscle. Induced contractile activity was shown to promote the differentiation of myotubes and results on directly stimulated, aneural myotubes indicated that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes. Moreover chronic electrical stimulation of primary myoblasts was shown to change and modulate myosin heavy chain expression depending on different impulse patterns [40, 41]. Another study conducted by Dennis et al compared the excitability and contractility of three-dimensional skeletal muscle constructs, engineered from C2C12 myoblast and 10T1/2 fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult rats. Created myooids were 12 mm long, with diameters of 0.1-1 mm, and were excitable by transverse electrical stimulation, and contracted to produce force. After approximately 30 days in culture, the specific force generated by the myooids was 2-8% of that generated by skeletal muscles of control adult rodents . Besides it has been shown that electrical stimulation of murine skeletal muscle cells enhances the expression of the angiogenetic factor VEGF and in vivo studies revealed that after 5 days of stimulation blood flow increased significantly . This is an interesting finding for the development of a tissue engineering approach with regard to provide functional muscle tissue in a clinical scenario, since vascularisation of tissue constructs is therefore an important prerequisite. The composition of the extracellular matrix (ECM) plays also a key role in the alignment and differentiation of myoblasts. The ECM should provide a framework for cell adhesion and tissue growth, which includes cell proliferation and differentiation. The matrix must be biocompatible and should be bioresorbable [22, 24, 44, 45]. The matrices used in tissue engineering are divided into synthetic and biologically-derived biomaterials . Saxena and collegues used polyglycolic acid (PGA) meshes seeded with myoblasts to transplant cells in vivo. After 6 weeks a vascularized muscle like tissue could be noticed . Other investigators established in vitro cell cultures cultivating muscle cells on Matrigel® [37, 50]. However Matrigel®, an extract from the Engelbreth-Holm-Swarm mouse sarcoma, contains various extracellular matrix proteins and growth factors in undefined concentrations. Besides it has the ability to change gene expression in cells . This matrix has been used in combination with collagen as a 3 dimensional scaffold, but in our opinion due to its origin its utility is limited for experimental models and not appropriate for a clinical setting. Various other biomaterials including collagens and alginate hydrogels have been used to replace the ECM in vitro, either to enhance the attachment of myoblasts or to alter their growth [24, 29, 44, 46, 51]. However, these matrices are not biodegradable and some are potentially immunogenic [46, 47]. Dennis and Kosnik introduced a way of designing a three dimensional skeletal muscle without using a primary matrix to provide a structural growth scaffold [28, 42]. They developed skeletal muscle tissue constructs by coculture of myoblasts and fibroblasts. The fibroblasts formed an extracellular matrix which was surrounding the myotubes. Furthermore Borschel and co-workers recently produced engineered skeletal muscle using an acellularized mouse extensor digitorum longus muscle as a scaffold. C2C12 myoblasts were injected into the acellular muscle matrix and isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation. Electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle . Since in vitro skeletal muscle tissue engineering involves culturing isolated primary myoblasts in an environment leading to the formation of a three-dimensional tissue construct, ideal matrices for such an approach should provide a high surface area for cell-matix interactions, sufficient space for extracellular matrix generation, and a minimal diffusion barrier during in vitro culture [53, 54]. Moreover, the matrix should be resorbable once it has served its purpose of providing a primary structure for the developing tissue . In many studies fibrin has been shown to provide these basic conditions as an ideal cell culture matrix: it is biocompatible and biodegradable [31, 54, 55] and consists of key-proteins of the ECM. Since cellular growth and differentiation depend on a structured environment which the cells need to interact with, fibrin supports the migration capacity of cells, allows the diffusion of growth and nutrition factors and has a high affinity to bind to biological surfaces . These properties are basic features of the hybrid skeletal muscle tissues which were developed by the authors: the incorporation of the myoblasts into a three-dimensional fibrin matrix . In our studies a high proliferation rate of the primary myoblasts within the fibrin-matrix could be observed, indicating the feasibility of fibrin as 3-D matrix. In order to evaluate the designed skeletal muscle tissue, we focused on myogenic transcription factors like MyoD and myogenin, and development of the acetylcholin receptor. We could show that myoblasts can proliferate and fuse to myotubes in the three dimensional fibrin matrix. Thus in our culture model the fibrin 3-D matrix was obviously the structural basis and the promotor of cell survival, proliferation and cell differentiation. Moreover, we established a co-culture system with neuronal slices of the spinal cord and myoblast in a three dimensional fibrin matrix. The results of our study confirmed that obviously the presence of a three-dimensional environment and of neuronal tissue is required for the understanding of the control mechanisms which are essential for in vitro regenerating of highly differentiated skeletal muscle tissue . However, aside from the induction of the differentiation of muscle cells, issues such as vascularization and innervation of in vitro generated muscle tissue constructs have to be more addressed, to provide functional muscle tissue in large volumes for clinical applications [5, 57].