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Review article

Engineering skeletal muscle tissue in bioreactor systems

An, Yang; Li, Dong

Author Information
doi: 10.3760/cma.j.issn.0366-6999.20141076

Abstract

Since increasing life expectancy, muscle diseases including skeletal myopathies such as muscular dystrophy or spinal muscular atrophy;1,2 in addition, aggressive tumor ablation, traumatic injury, and muscular atrophy, particularly in the elderly due to prolonged healing time caused by bone fractures and joint replacement because of arthritic diseases and prolonged denervation, are common clinical situations that often lead to significant loss of muscle tissue and need subsequent reconstruction procedures. These conditions are often correlated with major structural defects which may require clinically relevant treatments. However, the common autologous muscle grafting is associated with several potential problems, for example, the limited availability of muscle graft and the risk of donor site morbidity that result in volume deficiency and functional loss. Therefore, alternative strategies for skeletal muscle reconstruction are required. Tissue engineering (TE) represents a scientific approach that applies the principles of biology and engineering to the development of functional substitutes for structural defects.3 The field of TE focusing on the replication of neo-organogenesis has developed greatly over the past two decades.4 A great deal of researches have been performed to reconstruct various musculoskeletal types of tissue (e.g., bone,5 cartilage,6 and muscle7) and others (e.g., skin,8 cardiac valve,9 retina,10 vocal fold,11 liver,12 and nerves13). However, muscle TE has limitations and challenges. In order to engineer muscle tissue successfully, it may be beneficial to mimic the in vivo environment of muscle through application of adequate stimuli. Besides, the engineered muscle tissue must be vascularized and innervated for future clinical use. The purpose of this study was to give a concise review of the current state of art in TE of skeletal muscle, different kinds of dynamic bioreactor-based cultivation systems, and then significance for future clinical application.

Skeletal muscle tissue characteristics

Skeletal muscle consists of striated muscle tissue under control of the somatic nervous system. Its individual components, the muscle fibers,14,15 are formed by fusion of several undifferentiated immature cells, named myoblasts, into long, cylindrical, multinucleated cells during development. Owing to the arrangement of cytoskeletal elements in the cytoplasm of the muscle fibers, skeletal muscle exhibits a distinctive banding pattern. Myosin and actin, which are arranged in repeating units called sarcomeres, are the principal cytoplasmic proteins. A sarcomere is an arrangement of the contractile proteins myosin and actin, which form the thick and thin filaments, respectively. Myofibers are well-vascularized and multinucleated and derived from mesodermal myoblasts. After muscle injuries, myofibers become necrotic and are removed by macrophages.16 Mature muscle fibers are not capable of self-renewal due to terminal differentiation. The self-renewable ability of skeletal muscle tissue arises from a subpopulation of cells called satellite cells.17 According to histological description, satellite cells are undifferentiated cells residing between the sarcolemma and the basement membrane of muscle fibers, distinguishable from them by expression of Pax7. They can migrate to areas of injury through the basal lamina sheets, differentiate into myoblasts and fuse with preexisting, damaged fibers or with each other, and ultimately differentiate to muscle fibers.

Searching for the right source for engineering muscle tissue

As stated above, cells and extracellular matrix (ECM) have an inherently close and dependent relationship. Therefore, the type of cells and source of cells used to repopulate an organ-specific three-dimensional ECM scaffold are critical to the eventual functionality and clinical success of the engineered construct. Engineering a complex tissue or organ requires rebuilding parenchyma, vasculature, and underlying support structures, all of which differ in cell number and cell type depending on the organ of interest. An ideal cell would be one that can proliferate or self-renew as needed and yet give rise to the heterogeneous types of cells necessary to form a functional organ or tissue. Except in isolated cases where specific organ-derived differentiated cell types can be isolated, expanded in vitro, and used, the most likely candidate to fulfill such demands is a stem or progenitor cell. To truly build an organ or tissue requires generating the specialized structures of that tissue as well as any vascular or ductal components and any supporting structures replete with cells, including populations of resident stem or progenitor cells for ongoing organ maintenance.

As an alternative approach to host muscle transfer, muscle TE in vitro harbors promise for the treatment of skeletal muscle defects.18,19 Generally, most muscle TE approaches rely on the regenerative capability of the satellite cells and their potential for proliferation and differentiation.20,21 Myogenic satellite cells which are easily accessible and proliferate in vitro can be obtained from muscle tissue. Unfortunately, the differentiation processes toward myofibers still appear to be difficult to induce and control in vitro. To address these problems many studies concerning in vitro generation of muscular tissue made use of cell lines such as C2C12, which was a confirmed cell line of satellite cells from skeletal muscle of C3H mouse.22-25

Other cell types that have been demonstrated to differentiate into the myogenic cell line are hematopoietic stem cells (HSCs),26 embryonic stem cells (ESCs),27 and induced pluripotent stem cells (iPSCs).28 HSCs were investigated in damaged and uninjured skeletal muscle and it was demonstrated that HSC engrafted into damaged muscle tissue assigned to the restoration. The number of cells that were able to “home” appears to depend on muscle type and severity of muscle injury.26

ESCs isolated from the inner cell mass of a blastocyst can cause cell lineages of all three disparate germinal sheets and the germ lane and are thereby regarded as pluripotent.29 ESCs in company with myoblasts have come into the field of muscle TE recently.30 Adult fibroblasts can be transferred into pluripotent stem cells (PSCs) which can differentiate into all three germ layers in vitro but may cause teratoma in vivo.28 Thus, as an alternative source, iPSCs are comparable to ESCs concerning cell proliferation and pluripotency, but without the ethical problems that are implied with the use of ESCs. Although iPSCs have already validated their potential for regenerative medicine,31-34 research efforts made on iPSCs are still in their early stages and many difficulties have to be addressed before clinical practices are conceivable.35

As an alternative, mesenchymal stem cells (MSCs) have been proposed for muscle TE.36 MSCs as a possible cell source for muscle TE have distinct advantages compared to primary satellite cells. They can replicate repetitively without losing their differentiation capacities in early passages in contrast to primary myoblasts. Therefore, higher cell numbers may be generated out of a small population. Moreover, they can be transplanted in an autologous fashion and could have immunomodulatory functions in terms of promoting tissue regeneration rendering them attractive candidates for muscle TE.37,38 Populations of MSCs have been derived by various sources, while foremost sources described for muscular TE were umbilical cord blood,39 bone marrow,40,41 and adipose tissue.42,43 Bone marrow MSCs (BMSCs) have a high propagable capacity and are capable of self-renewal. This renders them suitable as progenitor cells for skeletal muscle TE.

Although BMSCs have been universally used, adipose-derived stem cells (ASCs) constitute an interesting alternative since they are easily accessible and reveal higher proliferation rates than BMSCs.44,45 ASCs have also been reported to have myogenic capability under special culture condition.46 These cells, which can be obtained by minimally invasive procedures from subcutaneous fat, are highly expandable in culture and can be readily induced to differentiate into muscle tissue-forming cells by exposure to a well-established myogenic medium. Adipose tissue is routinely available in liter quantities from patients undergoing liposuction, a minimally invasive procedure. After expansion, the yield of ASCs is relatively high, averaging about 200 000 cells per milliliter of lipoaspirate tissue.47

Extracellular matrix as a biologic scaffold

Biologic scaffold materials composed of ECM are commonly used to facilitate the constructive remodeling of a variety of tissues both in preclinical animal studies and in human clinical applications. The ECM used to create these scaffold materials is harvested from many different tissues. Nowadays, the ECM materials are harvested and typically processed as two-dimensional scaffolds and do not require anastomosis directly to the recipient vasculature for their clinical applications. Rather, the infiltrating or seeded cell populations rely on oxygen and nutrient diffusion for survival while a supporting vascular network develops over time. The composition of ECM is represented by a complex mixture of functional and structural molecules that affect a variety of cell activities. These molecules are arranged in a unique, tissue-specific, three-dimensional ultrastructure and are ideally suited to the tissue or organ from which the ECM is harvested. Therefore, the composition as well as the macroscopic and ultrastructural characteristics of three-dimensional, organ-derived biologic scaffold materials is highly complex. If properly prepared, such ECM scaffold materials can provide important microenvironmental cues necessary to support cell attachment, proliferation, and differentiation, while providing appropriate biomechanical support. These scaffold materials are in a constant state of flux and can be considered as temporary inductive siteappropriate templates to support the growth, differentiation, and function of the parenchymal cell population of each organ.

Vascularization and innervation of tissue-engineered constructs - indispensable ingredients for muscle engineering

Engineering a vascular bed inside a tissue construct remains a milestone of TE since it would allow the improved oxygen and nutrient exchange required to produce voluminous tissues in vitro and the immediate association of the tissue graft to the recipient circulation to prevent hypoxia-induced cell damage after transplantation. As skeletal muscle is a complex tissue with a high energy and oxygen demand, it seems to be evident that constructs require vascularization for clinically relevant practices. This is very significant, since a major limiting factor in generating skeletal muscle via TE is that myoblasts are unable to propagate and differentiate further than 150 μm away from a nutrient source and oxygen supply.48 Thus, every muscle construct must be connected to a vascular system in order to efficiently transport oxygen, carbon dioxide, and nutrients and waste products.

Tanaka et al,49 who developed an in vivo model of angiogenesis by creating an encapsulated microvascular arteriovenous shunt loop in the groin of rats, demonstrated the first results of generation and vascularization of autologous tissue constructs. Implantation of muscle constructs in vivo induces a foreign body reaction that gives rise to ingrowth of a supportive capillary network that promotes and maintains viability. Subsequently, Messina et al50 analyzed the interactions and potential of the AV loop chamber model in the formation of neotissue when a variety of different forms of skeletal muscle were inserted in their angiogenic model as an inductive source. Borschel et al51 who utilized the femoral vessels as axial pedicle generated vascularized functional skeletal muscle tissue in rats derived from primary myoblast cultures which produced a longitudinal contractile force when electrically stimulated. Using fibrin gel fixed around a preformed ectopic arteriovenous loop in rats, Bach et al52 also demonstrated that axial prevascularization seems to promote myoblast survival and kept their myogenic phenotype while not resulting in dedifferentiation of myoblasts. However, Levenberg et al30 first reported the neovascularization of in vitro tissue-engineered skeletal muscle. They perceived enhancement of the vascular network and developed the so-called prevascularized skeletal muscle constructs from cultures of myoblasts, endothelial cells, and embryonic fibroblasts. Further research is demanded to establish whether this model is transferable in humans. Furthermore, the addition of angiogenic factors may facilitate vascularization of the engineered tissue grafts. For instance, the use of genetically modified myoblasts expressing vascular endothelial growth factor (VEGF) has been investigated to enhance neovascularization and tissue mass of in vivo engineered muscle.53 In order to enhance functional integration of engineered skeletal muscle grafts, Koffler et al.54 described a first-time analysis of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo.

In contrast to the extensive research concerning vascularization of muscle constructs, there has been limited research about innervating tissue-engineered skeletal muscle constructs. A number of previous in vivo studies have demonstrated muscle atrophy after long-term denervation and the possibility to partially reverse this effect by chronic electrical stimulation (ES).55-57 It is important to comprehend the effect of chemotropic, electrical, and mechanical stimulation on cultured myoblasts related to their proliferation and differentiation. In co-cultures of myotubes and neural cells, neuromuscular-like junctions appear to form spontaneously. These nervehypher muscle constructs show spontaneous contractions and can also be stimulated.58 Larkin et al59 indicated in vitro that stimulated nerve-muscle constructs had better contractility characteristics than muscle-only constructs. Dhawan et al60 also showed that in vivo neurotization of skeletal muscle constructs caused better contractibility ex vivo than nonneurotized skeletal muscle constructs60 (Figure 1). In particular, coculturing muscle constructs with neural cells59,61 or inducing construct neurotization using transected nerves60 not only increased the differentiation and forced generation of muscle cells but also formed acetylcholinesensitive neuromuscular junctions and allowed indirect muscle stimulation via the neural extensions projecting from the tissue constructs. In addition, Bian et al62 used neonatal rat skeletal myoblasts cultured within three-dimensional engineered muscle tissue constructs that were treated with soluble recombinant miniagrin to promote contractile function of engineered skeletal muscle. Contractions could be elicited by electrically stimulating the neural extensions, although smaller forces are produced than with field stimulation. Further implantation studies with electrically stimulated or cocultured nerve-muscle constructs are expected to reveal the potential benefits of these approaches to in vivo innervation of engineered muscle tissues and their functional integration into the host muscle.

Figure 1. A:
Figure 1. A::
Drawing of a rat in supine position with left groin dissected, demonstrating the inguinal ligament, femoral artery, femoral vein, and femoral nerve. B: Drawing of a rat’s left groin with femoral nerve transected and sutured to the adventitia of the femoral artery. The silicone chamber filled with myoblast and fibrin gel has been placed around the vascular pedicle and proximal stump of the femoral nerve.

In addition, since stretching plays a key role in skeletal muscle tissue development in vivo, many groups attempt to investigate the effect of mechanical conditioning on the development of skeletal muscle engineered constructs. Candiani et al63 adapted cyclic mechanical stimulation to favor myosin heavy chain accumulation in engineered skeletal muscle constructs. Their preliminary findings showed that cyclic stretching induces myosin heavy chain (MHC) accumulation and contributes to myotube maintenance in a three-dimensional environment. Furthermore, ES is also required for the maturation of skeletal muscle and as a way to nondestructively monitor muscle development. ES can enhance the regenerative capacity of axotomized motor and sensory neurons. The contraction of myotubes is induced using outward current pulses and depends on the frequency.64 Yamasaki et al65 evaluated the relationship between myotube contraction and electrical pulses, investigated the effect of electrical pulse frequency on the excitability of myotubes, developed bio-actuators made of tissue-engineered skeletal muscle and suggested that a tissue-engineered bio-actuator may be controlled using electrical signals. Additionally, magnetite cationic liposomes were also used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs.66,67

Training the muscle - bioreactor systems used for tissue engineering

Bioreactors (Figure 2) are essential in TE, not only because they supply an in vitro environment simulating in vivo conditions for the growth of tissue substitutes, but also because they enable systematic investigations of the responses of living tissues to manifold mechanical and biochemical signals.

Figure 2. A:
Figure 2. A::
Schematic of spinner flask bioreactor. Scaffolds are suspended in culture while media is circulated using a stir bar. B: Schematic of rotating wall bioreactor. Outside wall of bioreactor rotates to circulate media. C: Schematic of perfusion bioreactor. Media is directly perfused through porous scaffold sealed into a growth chamber.

The modern utilization of bioreactors to grow muscle tissue in vitro is mostly based upon an earlier and much more abundant scientific literature on bioreactors just coming from physical adaptations of existing systems already developed for other tissue systems, for example, bone.68 In this section, bioreactor-based concepts for TE are summarized. Systems like spinner flasks using hydrodynamic shear stress, as well as rotating and perfusion bioreactors are introduced. Moreover, the concept of in vivo bioreactors and other novel bioreactors related to muscle TE are explained.

Spinner flasks bioreactor

The spinner flask is a simple and inexpensive bioreactor system. Convective forces are offered by a stirrer and medium flows around tissue. The whole system is placed in an incubator which controls temperature and oxygen content. Scaffolds are attached to a needle connected to the lid of the flask. Two angled side arms equipped with filters guarantee oxygenation of the medium. The degree of shear stress is associated with the stirring speed. A stirring rate of 50 rpm was used in a study with collagen and silk scaffolds seeded with human MSCs. It is obvious that cultivation of cartilage and cardiac constructs in spinner flasks generates engineered tissues that are superior to those cultivated under static conditions due to its well-mixed environment around the cells.69,70 However, minor modifications of the system have to be carried out by customers to ensure proper attachment of the scaffolds within the flask. This type of bioreactor is also used as a dynamic cell seeding device. Besides the beneficial effects with respect to differentiation and proliferation, another advantage of the system is its low cost of acquisition. A disadvantage of cultivating cell/ scaffold constructs in a spinner flask system is the possible formation of a dense superficial cell layer, which may have a very harmful effect on oxygen and nutrient supply of the cells in the center of the scaffolds.71 Furthermore, shear stress with the highest level on the bottom of the vessel in proximity to the stirrer leads to nonhomogenous conditions within a spinner flask.

Rotating bioreactor systems

Another system used in TE to enhance media mixing is the rotating wall bioreactor provided with two concentric cylinders, an inner cylinder that is immovable and supplies for gas exchange and an outer cylinder that rotates. The space between the two cylinders is entirely filled with culture medium and cell including scaffolds are placed freely moving in this space. The free locomotion of the scaffolds gives rise to a microgravity environment whereas the flow of the fluid, resulting from the centrifugal forces of the cylinder, balances with the force of gravity.72 Rotating wall vessel (RWV) devices were originally designed by the National Aeronautics and Space Administration to mimic microgravity.73-75 The laminar flow of a rotating vessel along a horizontal axis produces low levels of shear stress which are efficient to decrease diffusional limitations of nutrients and waste products. It is possible to grow relatively large three-dimensional clusters of all types of tissue including muscle76-79 which in some cases appear to be organized into tissue-like structures by using microcarriers which are suspended by the rotation of a cylindrical culture vessel on a horizontal axis. Nevertheless, due to differences in the structure of skeletal muscle compared to hollow organs including smooth or cardiac muscle rotating bioreactor systems appear to be more suitable for the growth of smooth and cardiac muscle than for skeletal muscle. Accordingly, it has been demonstrated that metabolic activity of engineered cardiac tissues grown in the RWV was superior to those of comparable tissues grown in static vessels.77 Other examples of successful usage of RWV include the culture of a retinal cell line to form three-dimensional retina-like structures,80 bone tissue,81 cartilage tissue,82 and tissue comparable to the temporo-mandibular joint disc.83 One disadvantage associated with the RWV is that growth of tissue is usually nonuniform.73 The centrifugal force leads to frequent collisions of the scaffolds with the bioreactor wall, which is a confounding factor that induces cell injury and disrupts cell attachment and matrix deposition on the scaffolds.84 Although rotating wall bioreactors are another relatively simple system that has shown effectiveness in some examples, perfusion systems have been indicated to provide a greater advantage in TE.

Perfusion-based bioreactor systems

Spinner flasks and rotating wall bioreactors do not effectively perfuse media into a scaffold. Bioreactors using a pump system instead to perfuse media directly through a scaffold are referred to as perfusion bioreactors. Perfusion bioreactors have two advantages compared to the systems described before: first, to circulate the culture media in order to provide enhanced mass transfer 86; second, to induce tissue organization and development by using the fluid dynamic forces. Many different perfusion bioreactor systems have been developed, but the majority of systems consist of a similar basic design containing a media reservoir, a pump, a tubing circuit, and a perfusion cartridge.86 The flow of medium through scaffold pores is favorable for cell differentiation promoting nutrient transport into the inner scaffold and providing mechanical stimulation in the form of liquid shear.87 Perfusion bioreactors have been mainly employed for osteochondral composites88 but have also been used for TE of functional muscle tissue89-92 with slight differences in reactor configuration. It must be taken into concern, however, that unlike bone, muscle does not appear to respond to fluid shear stress as an anabolic stimulus. In contrast, due to excessive shear stress, levels of flow rate exceeding a specific extent have shown to enhance the washing out of cells.93 This brings into question the need for dynamic fluid forces in the formation of mature skeletal muscle. Without the need for fluid dynamic forces, the main goal of muscle tissue perfusion is improved delivery of nutrients and removal of waste. In addition, the composition, morphology, and mechanical properties of engineered tissues grown in mechanically active environments are generally better compared to those grown in static environments, presumably due to increasing mass transport at tissue surfaces.73 Although transport limitations may not be an important problem in the engineering tissue, the distributions of cells and ECM in non-perfused engineered tissues are generally nonuniform. In native tissues, this problem is minimized by vascularization as well as loading-induced interstitial flow of fluid. In bioreactors, the interstitial flow of culture medium can be utilized to alleviate concentration gradients and thereby avoid the nonuniformities in tissue structure and function. Additionally, although it is obvious that the advantages of the utilization of perfusion-based bioreactor systems in TE, namely improved cellular proliferation, distribution, differentiation, and viability in the interior of scaffolds, outweigh those under static cultivation, there are some disadvantages compared with rotating wall bioreactor. In the perfused constructs mechanical stimulation importantly affected cell morphology, increased the incidence of cell proliferation, and reduced apoptosis. However, ECM deposition, cytoskeletal organization, and mechanical properties were poor. In rotated constructs cell proliferation was also higher and apoptosis lower than in static controls. Rotated constructs showed the highest ultimate stress and the lowest elastic modulus. Data indicated that the rotating bioreactor was more efficient than the perfusion bioreactor.

In vivobioreactor system

Except for in vitro bioreactor systems described above, the body can also be regarded as the ultimate bioreactor.94,95 The purpose of this approach for skeletal muscle TE is to reconstruct functional tissue through the cultivation and expansion of satellite cells in vitro followed by reimplantation using a transport matrix, which permits subsequent differentiation of cells in vivo. For many applications, many advantages of the use of the body as a bioreactor outweigh TE approaches ex vivo or in vitro. In particular, when autologous cells and tissues are used, it greatly simplifies the entire process so as to avoid tissue rejection from a transport matrix.

There are several instances of success with this approach in muscle TE. Okano et al96 reported that grafts comprising three hybrid tissues were inserted into the subcutaneous spaces on the backs of nude mice which were euthanized at 1, 2, and 4 weeks after the implantation. Four weeks after implantation a dense capillary network was formed in the vicinities and on the surfaces of the grafts. In the peripheral portion of the graft, multinucleated myotubes near rich capillaries were observed. Subsequently, Birla et al97,98 described a method of engineering contractile three-dimensional cardiac tissue with the incorporation of an intrinsic vascular supply and demonstrated the in vivo survival, vascularization, organization, and functionality of transplanted myocardial cells. Borschel et al99 also demonstrated that three-dimensional engineered skeletal muscle constructs produced longitudinal contractile force when electrically stimulated and can be engineered in vivo. The resulting tissues have histologic and functional properties consistent with native skeletal muscle. Furthermore, Dhawan et al60 also cultured rat myoblasts, suspended them in fibrin gel, and implanted within silicone chambers around the femoral vessels and transected femoral nerve of syngeneic rats for 4 weeks. Results indicated that neurotization of engineered skeletal muscle significantly increases force generation and causes neuromuscular junctions to develop, allowing indirect muscle stimulation. The ability of engineered muscle tissues to rapidly connect to the host neuromuscular system is expected to further facilitate their functional integration into the host environment and accelerate the functional recovery of the host muscle. Moon et al100 also described an in vitro preconditioning protocol that improves the contractility of engineered skeletal muscle after implantation in vivo. Primary human muscle precursor cells (MPCs) were seeded onto collagen-based acellular tissue scaffolds and subjected to cyclic strain in a computer-controlled bioreactor system. In vitro-engineered constructs were capable of generating contractile responses after 3 weeks of bioreactor preconditioning. This finding demonstrated cyclic mechanical preconditioning improves engineered muscle contraction.

Other novel bioreactors related to muscle tissue engineering

Except that systems using spinner flasks, and rotating and perfusion bioreactors, are introduced and the concept of in vivo bioreactors are discussed, some novel bioreactors relating to muscle TE are also introduced. Donnelly et al101 engineered an inexpensive muscle ES bioreactor to apply physiologically relevant ES patterns to tissue-engineered muscles and monolayers in culture. Subsequently, Barash et al102 described the features of a novel cultivation system, combining ES with medium perfusion for producing thick, functional cardiac patches. The models developed can be applied to different cultivation vessels, with different geometries. However, Candiani et al103 showed that cyclic stretching induces MHC accumulation and contributes to myotube maintenance in a three-dimensional environment, which will pave the way for effective engineered skeletal muscle construct development. Furthermore, Kensah et al104 described the design of a novel multimodal bioreactor for mechanical stimulation and real-time direct measurement of contraction forces under continuous sterile culture conditions. The bioreactor’s transparent cultivation chamber allows for microscopic assessment of tissue development including fluorescence imaging. Additional functions include electric pacing of tissues, as well as the possibility to perfuse the central cultivation chamber, allowing for continuous medium exchange and controlled addition of pharmacologically active agents. In addition, Clause et al105 developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue.

Conclusion and perspective

To date, various approaches have also been suggested to promote rapid vascularization and innervation of tissue constructs upon transplantation, which could have the benefit on long-term survival and functional integration into the host tissue. In addition, to optimize the cultivation of TE constructs, dynamic bioreactor systems, enhancing cellular proliferation and differentiation as well as addressing mass transport limitations, are appealing components. Muscle bioreactor systems include spinner flasks, RWV constructs, perfusion bioreactors, in vivo bioreactors, and other novel bioreactors related to muscle TE. These systems differ considerably with respect to ease of use, as well as monitoring and manipulation options. Currently available muscle bioreactors enable adequate monitoring and controlling of specific biological, physical, and chemical parameters during the process of in vitro muscle formation. Experiments utilizing perfusion systems have shown very promising results including induction of myoblastic differentiation.89

Ideally, a bioreactor-based muscle TE strategy would start with the extraction of a tissue or an organ with a vascular pedicle from a patient (Figure 3). This construct would then be uniformly placed in a bioreactor system. The population would be cultured within an environment that would result in the tissue or the organ to rapidly proliferate and undergo muscle differentiation with the bioreactor system monitoring oxygen content and automatically changing media when necessary. After a short but sufficient culture time, this construct would be removed and directly implanted in the patient where it would foster myoinduction and myoconduction for rapid repair of the defect site. With an enhanced amount of relevant animal studies and development of a clear strategy, bioreactor systems could play a key role in TE treatment of muscle defects and bring this strategy to clinical practice. Meanwhile, as the techniques of TE become more sophisticated, the usefulness of these methods for supporting the possibilities of reconstructive surgery will hopefully become reality.

Figure 3.
Figure 3.:
Ideal concept of tissue engineering presenting the basic principles of the technology.

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(Received September 9, 2014)

Edited by Cui Yi

Keywords:

tissue engineering; regenerative medicine; skeletal muscle; bioreactor; vascularization

© 2014 Chinese Medical Association