Clin Plastic Surg 30 (2003) 589 – 599

Engineering of muscle tissue
A.D. Bach, MDa,*, J. Stern-Straeterb, J.P. Beierb, H. Bannasch, MDb, G.B. Stark, MDb

Department of Plastic and Hand Surgery, University of Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany b Department of Plastic and Hand Surgery, University of Freiburg Medical Center, Hugstetter Str. 55, D-79106, Freiburg i. Br., Germany

Surgical reconstruction of muscle tissue lost by traumatic injury or tumor ablation is hampered by the lack of availability of functional substitution of native tissue. Until now, few alternatives have existed to provide functional restoration of lost muscle tissues. Free tissue transfer, although a common practice, is associated with significant donor site morbidity. Regenerating or engineering new tissues may one day allow the replacement of such lost, damaged, or failing tissues and organs. Techniques attempting regeneration of human tissues and organs (ie, tissue engineering) have recently entered into clinical practice. By continuing basic human and nonhuman embryonic and adult stem cell research, the future application of human stem cells (embryonic stem cells, adult stem cells, and progenitor cells) to generate replacement cells in clinical settings may become viable; this could help to avoid ethical issues and bypass problems of cellular rejection. Muscle tissues are categorized into three principle types: (1) skeletal muscle, (2) smooth muscle and (3) cardiac muscle. Each of these types is different in terms of anatomic structure, physiology, associated diseases, and potential to regenerate. This article reviews some of the recent findings resulting from tissue engineering science related to the contractile behavior of muscle tissue cells in a three-dimensional environment and discusses how tissue engineering could be used to create and regenerate skeletal,

smooth, and cardiac muscle. Even though these investigations have only recently been developed, numerous studies have been reported that indicate that these techniques may be of great importance in medicine in the near future.

Skeletal muscle tissue engineering Skeletal muscle is a highly specialized tissue. Its primary function is to generate longitudinal contraction. Skeletal muscles are unique tissues that 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 threedimensional matrix to form an organized tissue with high cell density and cellular orientation. These uniaxially structured cellular assemblages are characteristic of skeletal muscle tissue. During muscle injuries, myofibers become necrotic and are removed by macrophages [1]. Myoblasts (satellite cells) scattered below the basal lamina of myofibers, which remain in a quiescent and undifferentiated state, can enter the mitotic circle. This induces proliferation and fusion of myoblasts to form multinucleated and elongated myotubes that self-assemble to form a more organized structure, namely muscle fiber [2]. Satellite cells migrate and proliferate in the injured area and form a connective tissue network (muscle fibrosis). This process is called ‘‘scar tissue formation’’ and leads to a loss of functionality. The incidence of satellite cells in skeletal muscle is low (1% – 5%) and depends on age and muscle fiber composition [3].

* Corresponding author. E-mail address: Alexander.Bach@chir.imed. (A.D. Bach).

0094-1298/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0094-1298(03)00077-4

The concept of the in vitro tissue engineering approach. including skeletal myopathies (eg. The factors that play a major role in controlling the events leading to skeletal muscle development are MyoD. The implantation of engineered myoblasts has been used as a potential therapy for genetic muscle diseases such as Duchenne muscular dystrophy or for the repair Fig. and the antagonism between growth and differentiation [4.D. There are a variety of problems associated with the transfer of autologous muscle tissue to restore muscular function after tumor ablation. Implanted myoblasts might serve as vehicles for the delivery of recombinant proteins. . Therapeutic treatments for acquired and inherited skeletal myopathies require the ability to implant constructs of differentiated muscle tissue or to inject muscle or muscle-precursor cells into sites of dysfunction for subsequent formation of muscle tissue [11]. morphogenesis. A second approach involves the generation of satellite cells. the transferred tissue typically is not designed to function in its new capacity and may degenerate before integration into its new site. 2). as insulin like growth factor 1. 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 signaling systems controlling each developmental event. 1 and 2). or prolonged denervation [12]. which is a family of myogenic basic helix-loop-helix transcription factors [3. Moreover. expansion of cells in vitro. myogenin. such as angiogenic factors and growth factors. Therefore. 1. One way is to regenerate autologous satellite cells by biopsy. and vascular endothelial growth factor [13 – 15]. which allows differentiation into myotubes in vivo to occur (myoblast transfer therapy) (Fig. The common approach of transplanting muscle flaps is associated with donor site morbidity.6 – 8]. and reimplant the neotissue after differentiation has taken place (Fig. Duchenne muscular dystrophy and spinal muscular atrophy) and acquired muscle defects [9 – 11].5]. The construction of muscle tissue in vitro holds promise for the treatment of a variety of prevalent inherited human diseases. There are two general approaches to engineer artificial skeletal muscle tissue (Figs. and reimplantion of donor cells using a transport matrix. including mechanisms for cell differentiation. expand and differentiate cells in a three-dimensional defined environment in vitro in an artificial bioreactor. 1). myf-5. Surgical harvest of a significant mass of muscle causes functional loss and volume deficiency.590 A. erythropoietin. Bach et al / Clin Plastic Surg 30 (2003) 589–599 Skeletal muscle has become a general model for understanding many fundamental principles of development. trauma. and myf-6/MRF4/herculin. plastic and reconstructive surgery provides a wide field of indications in which tissue engineered skeletal muscle could be a promising approach.

it is necessary to investigate novel approaches for culturing functional. There have been several attempts to induce fusion of myoblast to myotubes in vitro. Human satellite cells can be successfully isolated and expanded in vitro from fetal and adult muscle biopsy [13. intracellular calcium-storage. In recent years. the differentiation process is difficult to induce. Therefore. are the ideal source for approaches in muscle tissue engineering. myoblast cell cultures need to be expanded to a great extent. and needs to be vascularized and innervated [13]. Few studies on the differentiation of myoblasts within a three-dimensional matrix have been reported. However. Although these techniques have been shown to improve the architecture and function of muscle as the myoblasts incorporate and differentiate within the target muscle. must integrate and regenerate lost muscle tissue. in vitro differentiation and maturation of satellite cells harvested from adult skeletal muscle could be an alternative source for treating muscular disorders [20]. primary cultures derived from satellite cells from rat myofibers grown in vitro have become the preferred source of myoblasts because they recapitulate muscle development more precisely than immortal myogenic cell lines [28]. This approach has many disadvantages because established cell lines approximate myogenesis less closely than primary myoblasts. Bach et al / Clin Plastic Surg 30 (2003) 589–599 591 Fig. cell lines such as C2C12 (which is an established cell line of satellite cells from skeletal muscle of C3H mouse) were used [20. with extending passaging of primary cells. Studies on the replacement of muscular tissues using tissue engineering methods have only recently started. The neotissue must be biocompatible.A. In many studies focusing on in vitro generation of muscular tissue. To create a tissue in vitro that can be reimplanted in vivo. donor cells must be autologous or nonimmunogenic [23]. and acetylcholine receptors. Important requirements of functional skeletal muscle are a parallel alignment of myofibrils with myosin/actin filaments. Therefore.24 – 27]. An understanding of the molecular control mechanisms of muscle development and differentiation is therefore an important prerequisite.29]. differentiated skeletal muscle tissue in vitro using primary myoblasts for autologous transplantation. which can differentiate into skeletal muscle cells. Mechanical stimula- . To obtain large volumes of tissue engineered skeletal muscle. primary satellite cells or other stem cells.21. imitating the in vivo conditions during myogenesis. they are limited by the large numbers of cells required and sites that must be injected [19]. of damaged myocardial tissues [16 – 18].D. To achieve this goal. which are needed for creating direct forces and functional use. The concept of in vivo tissue engineering approach. and living tissue substitutes for functional skeletal muscle replacement have not been developed successfully.22]. and many investigators have focused on the creation of functional muscle tissues in vitro [20. 2.

The extracellular matrix (ECM) content was significantly higher. including collagens and alginate hydrogels. Therefore.42. a dimeric growth factor that stimulates the migration and proliferation of endothelial cells. and the myofiber area percentage.35]. Chronic electrical stimulation of primary rat cells was shown to change the myosin heavy chain expression with different impulse patterns [37]. However. Matrigel. A force transducer measured passive forces and viscoelastic properties. excitability and contractility were measured from different cell types. Moreover. Other investigators established in vitro cell cultures cultivating muscle cells in Matrigel. to enhance the attachment of myoblasts. The ECM should provide a framework for cell adhesion and tissue growth.29]. the matrix should be resorbable once it has served its purpose of providing a primary structure for the developing tissue [44.50].592 A. Bach et al / Clin Plastic Surg 30 (2003) 589–599 tion is one important factor during myogenesis that influences gene expression. the in vitro regenerated skeletal tissue could achieve only 1% to 2% of forces of native skeletal muscle [29. cell number.35] and Kosnik [29] showed a way of designing a three-dimensional skeletal muscle without using a matrix to provide a structural growth scaffold. protein synthesis. and myofiber composition. Powell et al [32] created a mechanical cell stimulator that is able to stretch and relax the cell cultures in vitro. the tissue that resulted is not an appropriate substitute for functional implantation. which includes cell proliferation and differentiation. MA) as threedimensional scaffold by mechanical stimulation [32]. Dennis [34. these matrices are not biodegradable. Moreover. To improve the ratio of muscle fibers and ECM. a vascularized musclelike tissue could be seen [40].34. and total RNA/DNA content [30 – 32]. Saxena et al [40] seeded myoblasts onto polyglycolic acid (PGA) meshes and transplanted them in vivo.43]. It has the ability to change gene expression in cells [41] and promotes differentiation into myoblasts. This was due to a higher content of ECM and decreased muscle fiber content. and differentiation of myoblasts [24. myofiber density was low.48]. the elasticity. The matrices used in tissue engineering are divided into synthetic and biologically derived biomaterials. These conditions are essential for a matrix used in tissue engineering. Based on this background. They developed skeletal muscle tissue constructs by coculture of myoblasts and fibroblasts: The fibroblast formed an ECM that surrounded the myotubes. However. but because of its origin it is suitable only for experimental models and not for clinical use. The composition of the ECM plays an essential role in the attachment. myosin heavy chain expression during myogenesis can be modulated in vitro by electrical stimulation in cell cultures that consist of pre-differentiated skeletal muscle cells. The degradation rate can be modified by adding aprotinin. and some are potentially immunogenic [44. have been used to replace the ECM in vitro. Powell et al improved the development of three-dimensional human skeletal muscle tissue using collagen and Matrigel (Becton Dickinson. contains various extracellular matrix proteins and growth factors in undefined concentrations. although this neotissue comes closer to skeletal muscle than other attempts. and maturation was incomplete without stimulation [13. ideal matrices for such an approach should provide a high surface area for cell – matrix interactions.D. The mechanical stimulation improved the structure of the engineered skeletal muscle by increasing the mean myofiber diameter. The parallel alignment of myotubes can be induced by stretch stimulation [33]. The tissue engineered constructs from rats developed a mean of 1% of the specific force generated by the control adult rat muscle. The matrix must be biocompatible and should be bioresorbable. it is biocompatible and biodegradable and has a high affinity to bind to biologic surfaces [49. sufficient space for extracellular matrix generation.45]. It has been shown that electrical stimulation of murine skeletal muscle cells enhances the expression of the angiogenetic factor VEGF.47]. or to alter their growth [39. Using electrical stimulation. It has been used in combination with collagen as a three-dimensional scaffold. However. which is a . It has been shown that mechanical forces also have an important impact on mature skeletal muscle on myofiber diameter. and a minimal diffusion barrier during in vitro culture [46. Another approach of developing a higher differentiated and more functional skeletal muscle tissue is electrical stimulation. Fibrin possesses several important features as an ideal cell culture matrix: Because it is derived from serum preparations. Induced contractile activity promotes differentiation of myotubes [36]. After 6 weeks. which mimics the nerve stimulation during myogenesis and during regeneration of injured skeletal muscle. Various other biomaterials. and in vivo studies revealed that blood flow increased significantly after 5 days of stimulation [38]. alignment. Bedford. an extract from the Engelbreth-Holm-Swarm mouse sarcoma. Other studies focusing on the in vitro creation of skeletal muscle showed a different morphologic and functional appearance without mechanical stimulation in comparison to native skeletal muscle. Because in vitro skeletal muscle tissue engineering involves culturing isolated primary myoblasts in an environment leading to the formation of a three-dimensional tissue construct.39].

we focused on myogenic transcription factors. minimum cell toxicity. indicating the feasibility of fibrin as a three-dimensional matrix (Fig. Thus. . allows the diffusion of growth and nutrition factors. These properties are basic features of the hybrid skeletal muscle tissues that were developed by the authors (Fig. To evaluate the designed skeletal muscle tissue. The best results in terms of prolonged stability. In our culture model. 3). and cellular growth and differentiation depend on a structured environment that the cells need to interact with. (C) Phase photomicrograph of proliferated but undifferentiated mononucleated myoblasts in three-dimensional fibrin matrix after 10 days of culture. Different concentrations of fibrinogen and thrombin and seeding cell density in a three-dimensional matrix were evaluated. Myoblasts can proliferate and fuse to myotubes in the threedimensional fibrin matrix (Fig. myogenic specificity of the cells was evidenced by positive anti-desmin immunocytochemistry (Fig. and cell organization. and opacity (to enable evaluation by phase-contrast micros- copy) were obtained using aprotinin. Moreover. and different anti-fibrinolytic reagents were tested. After establishing a primary rat myoblast culture of high purity with an expansion through four passages.A. Fibrin supports the migration capacity of the cells. 3. 3D). (D) Immunostaining of primary myoblasts after co-cultivation with neuronal tissue for desmin. Three-dimensional monoculture. such as MyoD and myogenin. 3A – C). and is a nutrient medium [51 – 53]. proliferation.55]. we established a co-culture system with neuronal slices of the spinal cord and myoblast in a threedimensional fibrin matrix. showing development of myofibrils. and the results of our study confirm that a three-dimensional environment and neuronal tissue are required for the understanding Fig.D. the fibrin matrix serves not only as a threedimensional structure for the culture system but offers essential additional biologic properties. the fibrin three-dimensional matrix is the structural basis and the promotor of cell survival. The fibrin matrix consists of key proteins of the ECM. 3C). (B) Phase photomicrograph of proliferated but undifferentiated mononucleated myoblasts in three-dimensional fibrin matrix after 3 days of culture. and the acetylcholine receptor [54. A high proliferation rate of the myoblasts within the fibrin matrix could be observed. The pore structure of the fibrin can be adapted by different thrombin concentrations. (A) Phase photomicrograph of myoblasts in three-dimensional fibrin matrix on day 1. Bach et al / Clin Plastic Surg 30 (2003) 589–599 593 protease inhibitor.

depending on the microenvironment of the smooth muscle tissue [58. The replacement or regeneration of ischemic and infarcted areas of the heart could provide a potential therapeutic alternative to whole organ transplantation. the loss of cardiomyocytes leads to regional contractile dysfunction. and malignancy [63 – 65]. numerous investigators have attempted application of alternative materials and tissues for bladder. After injury. intestine. smooth muscle cells enter into a proliferative state and produce new ECM proteins [56. however. Myocardial infarction followed by heart failure is one of the major causes of morbidity and mortality. The replacement of defective cardiac tissue by a functioning myocardium offers an exciting option in cardiovascular medicine. proliferative stimuli for these cells include a wide range of growth factors. and on histologic examination all animals had normal cellular organization of the bladder with a urotheliallined lumen surrounded by submucosal tissue and smooth muscle. The engineered bladders were able to show normal function with time. and other exogenous factors. when cultured in vitro and cultured on collagen scaffolds. and the engineering of three-dimensional cardiac tissue constructs in vitro offers new perspectives for basic cardiovascular research and for tissue replacement therapy [73 – 77]. and increased expression of rough endoplasmic reticulum [67 – 70]. and bladder. This change involves the loss of contractile ability. assume a dedifferentiated or proliferative phenotype when placed in culture. Any effort to engineer these organs must address the formation of structurally and functionally appropriate smooth muscle tissue. mechanical forces. and the spontaneous formation of three-dimensional cell aggregates. issues such as vascularization and innervation of in vitro generated muscle tissue constructs have to be addressed to provide functional muscle tissue in a clinical scenario.594 A. similar to vascular smooth muscle cells. Smooth muscle tissue engineering Smooth muscle has the greatest regenerative capacity among the three types of muscle tissues. . Because of the problems encountered with the use of gastrointestinal segments. The animal model involved an initial biopsy of tissue from normal bladders with cell expansion in vitro and seeding them onto matrices. perforation. such as carbachol [67]. decreased contractile protein content. Atala et al reported on using allogenic collagen grafts and scaffolds based on PGA polymer for bladder augmentation in dogs [63. Bladder smooth muscle cells. and they respond to mitotic signals by cell hypertrophy rather than by cell hyperplasia. and ureter replacement.D. including infection. The signal transduction pathways that control the proliferation of the smooth muscle cells are not known in detail. urethra. such as blood vessels. The tissue grafts were prepared and preseeded with smooth muscle cells on one side and urothelial cells on the opposite side. For the repair of heart tissue to be effective. Bach et al / Clin Plastic Surg 30 (2003) 589–599 of the control mechanisms that are essential for in vitro regenerating of highly differentiated skeletal muscle tissue. Adult smooth muscle cells are quiescent and possess a contractile phenotype.59]. Dispersed vascular smooth muscle cells show rapid modulation from the contractile to the proliferative status.66]. Because adult mammalian cardiac muscle lacks stem cells. cardiomyocytes withdraw from the cell cycle and terminally differentiate [71.63].57]. metabolic disturbances. The suitability of engineered heart tissue for in vitro and in vivo applications depends on the degree of syncytoid tissue formation and cardiac myocyte differentiation in vitro. contractile function. However. The efforts of engineering tissues of the urinary tract had the most important impact on the research in the field of smooth muscle cells [60 – 62]. Further improvements in the use of previously described materials and cell seeding procedures and the development of new biodegradable and biocompatible materials combined with refinements of cell culture techniques may result in clinically successful bladder grafts for bladder augmentation and even tually even for whole bladder replacement and for the reconstruction of other tissues in the genitourinary tract [62. Thus. The bladders augmented with the cell-seeded grafts showed a 100% increase in capacity compared with a 30% increase in the nonseeded grafts. urolithiasis. Cardiac muscle tissue engineering Cardiomyocytes do not regenerate after birth. demonstrated a loss of contractile response to normal in vivo pharmacologic stimulators. and necrotized heart muscle cells in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissue [72].72]. little is known about the phenotypic and functional characteristics of cultured human bladder smooth muscle cells. The common approach of using bowel for genitourinary tract reconstruction is associated with a variety of complications. Bladder smooth muscle cells. Although these studies are promising. cardiac tissue engineering is an emerging field of research. Smooth muscle cells are an integral component of a number of tissues. Shortly after birth.

the future application of human stem cells (embryonic stem cells. several scaffolds of proteins and synthetically produced polymers have been tested. a cardiac phenotype. In most studies.76. devastating. (5) three-dimensional constructs lack adequate plasticity and mechanical stability for implantation purposes.91]. gelatin. especially matrix proteins. Morphologic. discovery of factors responsible for proliferation of adult cardiomyocytes. and the first results are promising [90]. The concept of expanding autologous skeletal myoblasts ex vivo and injecting them into the postinfarction scar during coronary artery bypass grafting has been transferred to humans by Menasche et al. studies of pluripotent mouse embryonic stem cells have led to in vitro models of cardiomyocyte differentiation. alginate. Cardiomyocytes cultured under conditions of oscillating tension align themselves parallel to the direction of force. myotubes must be orientated so that their contractile ability enhances the function of the myocardium. transcription factors. and suitable techniques of gene transfer might allow for the production of autologous artificial myocardium-like tissue that is capable of correcting myocardial injury and restoring impaired heart function. However. adult stem cells. and molecular techni- ques indicate that the in vitro maturation process recapitulates the developmental pattern of early cardiogenesis. This alignment of cardiomyocytes could play an important role in the organization of myotubes after implantation [78 – 81].D. However. there seem to be some principal problems that are associated with this approach: (1) Scaffold materials often exhibit an intrinsic stiffness that may compromise diastolic function.77 – 80]. Accordingly. at least in part.A.93]. the proof of direct participation of the grafted cells in overall cardiac contraction is lacking. Moreover. are not exactly defined or clinically approved or are not suitable for human use (eg. smooth muscle cells.78. Matrigel) [76. and (6) the materials used. An alternative approach to the above mentioned cell-grafting procedures may be the replacement of diseased myocardium tissue with in vitro-designed cardiac constructs [75. by continuing basic human and nonhuman embryonic and adult stem cell research. and the other uses in vitro-designed tissue equivalents [79. who demonstrated that stem cells.74. A most elegant approach has been used by Orlic et al. Two principle strategies for the replacement of impaired myocardium have been used. further progress in stem cell technology. In addition. Thus. despite the survival and differentiation of implanted cells. was rarely observed [84. and progenitor cells) in clinical settings may become viable [93]. Formation of scar tissue leading to structural alterations and inhibiting contact between grafted cells and host tissue seems to account for this problem. The vascularization of in vitro-engineered tissues might result in the generation of a complete bioartificial heart in the future [73. One approach uses the application of isolated cells [82 – 91] (see Fig. The effect seemed to be independent of cell origin because positive results were reported from fetal or neonatal cardiac myocytes. Immature cells from embryonic chicken and from fetal or neonatal rats seem to have the capacity to reconstitute tissue-like structures of different shapes and sizes when they are cultured within a scaffold substratum. ECM components. demonstrating the potential of an autologous adult stem cell approach. electrophysiologic. and pluripotent stem cells [82 – 91]. Bach et al / Clin Plastic Surg 30 (2003) 589–599 595 transplanted cells have to differentiate and to be electrically coupled to the host cardiomyocytes. fibroblasts. including collagen. The exciting aspect of this report was that the stem cells acquired. mechanical and electrical cell – cell contact between graft and host. 2). the injection of cells into the scar tissue improved global heart function. and genetic studies have shown how signaling molecules. and polyglycolic acid. (4) cellular distribution and viability are not homogeneous.80]. mainly in the cryo-injury or in the myocardial infarction model after coronary ligation in mice and rats. and therefore obtained three-dimensional constructs are too small to allow surgical implantation into infarction areas.80] (see Fig. Summary The loss or failure of an organ or tissue is one of the most frequent. and costly problems in . (2) biodegradation of the scaffold materials remains often incomplete. For in vitro tissue construction. Moreover. In summary. which is a main requirement for synchronous contractions. (3) size limitations of engineered constructs exist that are due to a lack of metabolic or oxygen supply in the core of the three-dimension constructs. Successful implantation of pluripotent stem cells into the infarction scar has been reported recently in mice [92]. transdifferentiated into cardiac myocytes after the injection of cytokines [85]. endothelial cells. liberated from bone marrow and transferred to infarcted myocardium. and calcium-handling proteins affect this process. it is controversial whether these effects are due to the ability of these cell types to create sufficient amounts of new myocardium-like tissue within the infarction area and whether the cells are able to participate in synchronized heart contraction. 1).

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