a Department of Plastic and Hand Surgery, University of Erlangen-Nürnberg, Germany b Department of Plastic and Hand Surgery, University of Freiburg, Germany
Received: August 18, 2004; Accepted: October 10, 2004 * Correspondence to: Alexander D. BACH, M.D. Department of Plastic and Hand Surgery, University of Erlangen Medical Centre,
D-91054 Erlangen, Germany. Tel.: ++49-9131-85-33277, Fax: ++49-9131-85-39327 E-mail: Alexander.Bach@chir.imed.uni-erlangen.de
The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution of this native tissue. Until now, only few alternatives exist to provide functional restoration of damaged muscle tissues. Loss of muscle mass and their function can surgically managed in part using a variety of muscle transplantation or transposition techniques. These techniques represent a limited degree of success in attempts to restore the normal functioning, however they are not perfect solutions. A new alternative approach to addresssing difficult tissue reconstruction is to engineer new tissues. Although those tissue engineering techniques attempting regeneration of human tissues and organs have recently entered into clinical practice, the engineering of skletal muscle tissue ist still a scientific challenge. This article reviews some of the recent findings resulting from tissue engineering science related to the attempt of creation and regeneration of functional skeletal muscle tissue.
Keywords: tissue engineering • skeletal muscle • cell culture • myoblasts • satellite cells • myopathies
Source: J. Cell. Mol. Med. Vol 8, No 4, 2004 pp. 413-422
Tissue engineering represents a scientific approach that attempts to mimic neoorganogenesis . The creation of skeletal muscle tissue using tissue engineering methods holds promise for the treatment of a variety of muscle diseases, including skeletal myopathies such as muscular dystrophy or spinal muscular atrophy [2, 3]. In addition traumatic injury, aggressive tumor ablation and prolonged denervation are common clinical situations that often result in significant loss of muscle tissue requiring subsequent surgical reconstruction. Until now, only few alternatives exist to provide functional and aesthetic restoration of lost muscle tissues aside from transfer of muscle from local or distant sites. However, this technique of free tissue transfer, although a common practice, is associated with significant donor site morbidity causing functional loss and volume deficiency. Therefore plastic and reconstructive surgery also provides a wide field of indications, in which tissue engineered skeletal muscle could be a promising approach  . Moreover regenerating or engineering new tissues may be a potential solution for the replacement of lost, damaged or failing tissues and organs in general [1, 6]. Even though these investigations have only recently been developed those techniques attempting regeneration of human tissues have recently entered into clinical practice in the case of tissues such as skin, bone or cartilage [7–12]. However the engineering of skletal muscle tissue remains still a challenge. This article reviews some of the recent findings resulting from „tissue engineering science“ related to the differentiation of muscle tissue cells within a three dimensional environment and discusses how tissue engineering techniques could be introduced to create and to regenerate skeletal muscle tissue.
Skeletal muscles are composed of bundles of highly oriented and dense muscle fibers, each a multinucleated cell derived from myoblasts. The muscle fibers in native skeletal muscle are closely packed together in an extracellular three-dimensional matrix to form an organized tissue with high cell density and cellular orientation to generate longitudinal contraction. After muscle injuries, myofibers become necrotic and are removed by macrophages . A specialized myoblast sub-population called satellite cells scattered below the basal lamina of myofibers are capable of regeneration . The incidence of satellite cells in skeletal muscle is very low (1%–5%) and depends on age and musclefiber composition . These cells remain in a quiescent and undifferentiated state and can enter the mitotic circle in response to specific local factors . This induces proliferation and fusion of myoblasts to form multinucleated and elongated myotubes, which self-assemble to form a more organized structure, namely muscle fiber . Besides satellite cells migrate and proliferate in the injured area and can form a connective tissue network (muscle fibrosis). This process is called “scar tissue formation“ and leads to a loss of functionality [16, 17].
The engineering of muscle tissue in vitro holds promise for the treatment of skeletal muscle defects as an alternative to host muscle transfer [1, 3, 4, 18, 19]. Skeletal muscle tissue engineering depends on the regenerative properties of the satellite cells and their potential for proliferation and differentiation, since these primary skeletal muscle cells can be harvested from adult muscle and successfully grown in vitro [15, 20]. Important requirements of engineering functional skeletal muscle are a parallel alignment of myofibrils with myosin/actin filaments, intracelluar calcium-strorage and acetylcholin receptors, which are needed for creating direct forces and functional use. Besides the neotissue must be biocompatible, needs to be vascularised and finally needs to be innervated [5, 21]. In order to obtain large volumes of tissue engineered skeletal muscle, myoblast cell cultures need to be expanded to a great extend. However, with extending passaging of primary cells, the differentiation process is difficult to induce. To overcome these problems in many studies focussing on in vitro generation of muscular tissue cell lines such as C2C12 which is an established cell line of satellite cells from skeletal muscle of C3H mouse, were used [22–25]. However this approach seems to have disadvantages, because established cell lines approxi-the replacement of muscular tissues using tissue mate myogenesis less closely than primary engineering methods have only recently started and myoblasts. Therefore primary cultures derived from many investigators have focussed on the creation of satellite cells from myofibers grown in vitro are the functional muscle tissues in vitro [23, 28, 29]. preferred source of myoblasts because they recapit-However, few studies on differentiation of ulate muscle development more precisely than myoblasts within a 3-D matrix have been reported immortal myogenic cell lines [5, 26, 27]. Studies on and living tissue substitutes for functional skeletal muscle replacement have not yet been developed successfully. To achieve this goal it is necessary to investigate novel approaches for culturing functional, differentiated skeletal muscle tissue in vitro using primary myoblasts for autologous transplantation. An understanding of the molecular control mechanisms of muscle development and differentiation is therefore an important prerequisite. It is becoming apparent that the circumstances related to the growth of cells in three-dimensional scaffolds in vitro are revealing aspects of the phenotypes of cells and insights into cell behaviors that would have otherwise escaped view. In this regard skeletal muscle tissue engineering has become a general model for understanding many fundamental principles of development, including mechanisms for cell differentiation, morphogenesis and the antagonism between growth and differentiation [30, 31]. Many of the steps involved in the development of myoblasts from mesodermal precursor cells and their subsequent differentiation into multinucleate muscle fibers correspond to the expression of specific transcription factors and signalling systems controlling each developmetal event. The factors which play a major role in controlling the events leading to skeletal muscle development are MyoD, myf-5, myogenin and myf-6/MRF4/herculin, a family of myogenic basic helix-loop-helix transcription factors [32-34]. The proper spatial and temporal expression of these transcription factors is critical for successful myogenesis .
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].
Therapeutic treatments for acquired and inherited skeletal myopathies and loss of functional muscle tissue require the ability to either the implantion of differentiated muscle tissue constructs or the injection of muscle-precursor cells into sites of dysfunction or tissue deficiency for subsequent formation of new muscle tissue  . The implantation of engineered myoblasts has been utilized as a poten
In contrast to these myoblast transfer strategies, other researchers in the field of muscle tissue engineering are more focussing on in vitro differentiation and maturation of satellite cells harvested from adult skeletal muscle. This approach of in vitro development of bioartificial muscle could be an alternative source for treating muscular disorders as described above  . These attempts reflect the two general approaches to engineer skeletal muscle tissue. One approach uses in vitro-designed and pre-fabricated artificial muscle tissue equivalents to reimplant the neo-tissue after differentiation has taken place (in vitro tissue engineering) (Fig. 1A). The second approach uses the application of isolated satellite cells, after expansion of cells in vitro using an appropriate transport matrix, which allows differentiation into myotubes in vivo to occur (in vivo tissue engineering) (Fig.1B). Future developments and the decision regarding which approach is more promising depend on the elucidation of the relationships among cell growth and and differentiation, the cell integration capacity in the host in in vivo experiments and the capability to induce vascularisation of tissue equivalents in vitro.tial therapy for genetic muscle diseases such as Duchenne´s muscular dystrophy or for the repair of damaged myocardial tissues (Myoblast Transfer Therapy)   . The rationale behind this strategy is that the implantation of large numbers of myoblasts result in the fusion of these cells to the affected tissue, thereby improving the functional status of the muscle . Early results have demonstrated that exogenously introduced myoblasts are incorporated into local target sites and fuse with existing myofibers . However, this techniques, although shown to improve the architecture and function of muscle as the myoblasts incorporate and differentiate, is limited by the large numbers of cells required and sites that must be injected  . Nevertheless implanted and in vitro transfected myoblasts might serve as vehicles for the delivery of other recombinant proteins such as angiogenic factors and growth factors as insulin like growth factor 1, erythropoeitin and VEGF [21, 63–65]. This myoblast-targeted gene therapy with the potential for local production and release of needed therapeutic proteins holds promise for the treatment of several myopathies as well as other diseases [65–67], lacking important functional proteins [64, 68].
It has been shown that satellite cells can be successfully isolated and expanded in vitro from fetal and adult muscle biopsy . In order to create a tissue in vitro that can be reimplanted in vivo, donor cells must be autologous or at least nonimmonogenic . Therefore, primary satellite cells or other stem cells, which can differentiate into skeletal muscle cells, are the ideal source for transplantation approaches in muscle tissue engineering. One major problem that cell transplantation studies imitating autologous transplantation are facing at the moment is the ability of detecting transplanted cells after integration in the host. Therefore we developed an approach to create functional skeletal muscle tissue in vivo using the transplantation of primary myoblasts precultivated within a three-dimensional (3D) fibrin matrix and to determine the fate of the transplanted cells using the Y chromosome detection technique in a syngeneic rat animal model . 3D myoblast cultures were established derived from male donor rats and after 7 days of cultivation we performed an orthotopic transplantation of 3D-cell constructs into a created muscle defect within the gracilis muscle of syngeneic female rats (Fig 2A). These transplanted cells showed a poitive desmin immunostaining, supporting the assumption of the cells retaining their myogenic phenotype and Y chromosome in situ hybridization indicated the survival and integration of transplanted male myoblasts into the female recipient animal (Fig. 2B). After 50 days male donor cells still could be tracked, now as Y chromosome positive nuclei incorporated into host tissue, resulting in several mosaic multinuclear muscle fibers, consisting of cells of male and female origin (Fig 2C,D). These fibers were restricted to the area around the implantation site of the 3D-cell construct, indicating that transplanting myoblasts in a syngeneic rat model results in regeneration of skeletal muscle fibers by incorporation of myoblasts into host myofibers (Fig. 2C,D). Thus this approach to tissue engineering, transplantation of a small number of cells and growing the tissue in vivo, may bypass current difficulties in the in vitro engineering of large tissue masses for subsequent transplantation that are related to the lack of sufficient vascularization. Moreover this approach may be ideal for the utilization of small numbers of stem cells in the regeneration of skeletal muscle tissue . In summary, further progress in stem-cell technology , as well as discovery of conditions responsible for the control-mechanisms of proliferation and differentiation of adult satellite cells, combined with suitable techniques of vascularization might allow for the production of autologous artificial skeletal muscle-like tissue that is capable of correcting muscle injury and restoring impaired muscle function.
Tissue engineering and regenerative medicine is an exiting interdisciplinary field, which applies to the principles of engineering and biology to the development of viable substitutes that restore the function of damaged tissues and organs. Until now the efforts of tissue engineering science have provided new knowledge that have deepen our understanding of the phenotype and behavior of the skeletal muscle cells. This knowledge may in turn enable promising advances in musculoskeletal tissue engineering. Growing new tissues (neoorganogenesis) is a complex process that requires the teamwork of developmental and cellulare molecular biologists, engineers, material scientistes, and physicians. As the techniques of tissue engineering become more sophisticated, the usefulness of these methods for supporting the possibilities of reconstructive surgery will hopefully become reality.
Fig. 1 A. The concept of in vitro - tissue engineering approach. B. The concept of in vivo - tissue engineering approach.
Fig. 2 Y chromosome in situ hybridization of transplanted myoblasts. Positive nuclei of syngeneic male donor rats indicated by arrows. A + B) 2 days after transplantation: fibrin matrix still present (host muscle fibers of gracilis muscle indicated by arrow heads, fibrin matrix is indicated by hollow arrow heads); C) 14 days after transplantation: male myoblasts fused with female host myofibers of the gracilis muscle; D) 50 days after transplantation: male myoblasts fused with female host myofibers of the gracilis muscle, resulting in several mosaic multinuclear muscle fibers, consisting of cells of male and female origin.
Source: J. Cell. Mol. Med. Vol 8, No 4, 2004 pp. 413-422