1.1. Has Churchill been right?
In 1932, Sir Winston Churchill predicted that it will be possible in future to grow and engineer muscle tissue
A frequent clinical application of skeletal muscle tissue is the microsurgical transfer of myocutaneous free flaps for the coverage of soft tissue defects. As one major disadvantage, the use of free flaps is inevitably linked with a certain morbidity at the donor site including the loss of functional muscle tissue. In this situation, engineered muscle tissue could help to reduce the donor site morbidity. Above all, the advantage of muscle TE lies in the generation of functioning muscle tissue to replace certain muscles after damage or denervation (Klumpp et al. 2010). For example, the treatment of facial nerve palsy is momentarily limited to the transfer of autologous muscle tissue innervated by another nerve (the trigeminus nerve, e.g.) or free transfer of distant muscle tissue (Terzis & Konofaos 2008). Though multiple techniques and modifications exist, the results yielded in those clinical situations are moderate (Kumar & Hassan 2002, Terzis & Noah 1997). Furthermore, Kim
However, a clinical application of skeletal muscle TE has not been realized to date due to certain obstacles which will be discussed in the following. Though,
2. Finding the matrix for muscle TE
A variety of materials has been analyzed and reviewed regarding their suitability for skeletal muscle TE. On one side, natural materials like collagen I in the first place are preferred due to their biocompatibility and their close resemblance to the natural extracellular matrix (ECM). On the other side, synthetic materials generally show greater stability as well as cost-saving and easy handling.
Regarding collagen I - the main component of the natural ECM of mature skeletal muscle tissue - its advantage lies in high elasticity which is a pre-requisite for muscle contraction. Thus, its mechanical properties
Biodegradable synthetic polymers have also been widely used for muscle TE. Their advantages lie in easy handling and very good stability
Taking the properties of the most frequently used biopolymers and synthetic polymers into account, the complexity of mature skeletal muscle tissue asks for a combination of different complementary materials to engineer a matrix that meets the special demands of skeletal muscle TE. Composite scaffolds containing both, synthetic polymers for stability as well as biopolymers for enhanced cell attachment and elasticity, are therefore preferred in muscle TE research. E.g. PCL has been combined with collagen (Choi et al. 2008), gelatine (Kim, M.S. et al.), PLLA (Engelhardt et al.) and other materials. Also the combination of silk fibroin with collagen leads to suitable mechanical properties with good cell attachment
Comparable to the wide variety of materials, the methods of processing different forms of matrices are equally numerous. Thereby, mechanical properties of a certain matrix, degradation rates and cell attachment depend on the scaffold’s architecture. Whereas single-cell-layers can be easily cultured on two-dimensional scaffolds (films, micropatterned scaffolds), the architecture of three-dimensional scaffolds is more complex. To ensure cell survival and proliferation
Concerning the pore size, hydrogels usually show freely diffusion of nutrients and oxygen. Cells incorporated in hydrogels can migrate through the scaffold by degrading the gel but at the same time the stability of the gel decreases continuously. Furthermore, the architecture of hydrogels randomly spread pores (Fig. 2). However, the natural extracellular matrix within functional skeletal muscle tissue is highly orientated.
The parallel alignment of ECM and skeletal muscle tissue is the pre-requisite for effective muscle contraction and force-generation along a longitudinal axis. Curtis and Wilkinson first described the “cell guidance theory” by demonstrating that microgrooved matrices with a parallel micropattern provoke parallel aligned cell growth along the pattern of the scaffold (Curtis & Wilkinson 1997). This phenomenon is also present in myoblast culture (Choi et al. 2008, Huang et al. 2006, Huber et al. 2007) facilitating the generation of aligned myotubes (Gingras et al. 2009). Therefore, several techniques for aligned scaffold architecture have been developed including selective laser sintering or three-dimensional printing reviewed by Karande et al (Karande et al. 2004). Another method to gain spatially orientated pores in sponge-like matrices is unidirectional freeze-drying of materials like collagen (Madaghiele et al. 2008) or silk fibroin (Mandal & Kundu 2009, 2009). Hydrogels are gradually frozen leading to controlled formation of ice crystals which result in controlled porosity after evaporation of the aqueous part of the hydrogel. Additionally, Schoof and co-workers demonstrated that also the pore-size can be controlled by variation of the freezing-temperature (Schoof et al. 2001). Though, there are certain disadvantages linked to the freeze-drying method. First of all, secondary surface modifications of the sponges like coating procedures to enhance cell attachment are difficult and sparsely controllable. Secondly, the alignment of the pores is only spatial whereas the architecture of the matrix surrounding the pores usually remains at random.
On the contrary, electrospinning as an alternative method offers strict alignment of the resulting scaffolds (Ayres et al. 2006). The technique of electrospinning results in fibers formed by electrical voltage (Boudriot et al. 2006). Though, the process depends on multiple parameters, e.g. concentration and viscosity of the spinning solution, the voltage applied during the spinning process or flow rate of the spinning solution. The mechanical and chemical properties of electrospun matrices can be adjusted to the demands of the respective tissue by varying these parameters. Thus, a variety of synthetic and biopolymers can be electrospun at the micro- or nanoscale (Sell et al. 2009). Nanofibrous matrices electrospun from ECM proteins such as collagen I or hyaluronic acid mimic the natural ECM exactly and therefore ensure excellent cell attachment, cell viability and differentiation (Barnes et al. 2007). As mentioned before, biopolymers often lack the suitable stability for
The more complex core-shell-spinning technique uses two separate polymers which are electrospun co-axially with the second polymer surrounding the first polymer at the core. Zhang
However, electrospinning of aligned nanofibrous matrices is linked with poor control of the pore size as the main disadvantage. The generation of electrospun three-dimensional matrices, especially, results in densely packed scaffolds (fig. 3) that hinder cell infiltration (Baker & Mauck 2007, Telemeco et al. 2005). Therefore, co-spinning of water-soluble sacrificial fibers such as poly-(ethylene-oxide) (PEO) has been shown to overcome this problem (Baker et al. 2008). The sacrificial PEO fibers are interspersed inside the three-dimensional matrix and dissolve easily in water and alcohol during sterilization procedure before cell seeding. The resulting interspaces between the residual fibers then enable cells to migrate through the matrix (Baker et al. 2008). Though the control of pore size and interspaces in orientated nanofiber matrices is still challenging, the electrospinning technique holds great potential for TE and regenerative medicine and therefore pretends to be the most promising matrix for skeletal muscle TE at the moment.
3. Cell source
3.1. The satellite cell
Satellite cells form the major source for muscle regeneration
This myogenic imprinting renders the satellite cell to be a safe cell source for
Recently, the existence of yet another cell population in adult skeletal muscle tissue has been proved: The telocyte was described by Popescu
3.2. Stem cells
Stem cells of different origin offer a unique proliferation potential as the main advantage. To date, adult stem cells play the most important role in TE research, though other sources exist (embryonic or induced pluripotent stem cells (iPSC) e.g.) (Klumpp
Though their poor incorporation into myofibers and – compared to satellite cells - less effective myogenic differentiation
4. Cell survival
in vivo/ vascularization
Whereas the generation of two-dimensional skeletal muscle tissue
Thus, vascularization in general as well as number and pattern of vessel ingrowth of different matrices can be analyzed (Arkudas et al. 2010, Polykandriotis et al. 2009) and the vascularized matrix offers a platform for tissue engineering for skeletal (Messina et al. 2005) or cardiac (Morritt et al. 2007) muscle
Depending on the matrix architecture, a certain period of time is necessary for vascularization of the whole construct. This pre-vascularization time plays an important role for survival rates of implanted cells
Beside angiogenic growth factors, other factors such as insulin-like growth factor-1 (IGF-1) have been shown to increase survival rates of implanted cells
5. Myogenic differentiation
5.1. Molecular factors for myogenic differentiation
Beside the improvement of cell survival rates, the myogenic differentiation of implanted muscle precursor cells into functional skeletal muscle tissue
The well-known factor IGF-1 has been demonstrated to increase the proliferation as well as myogenic differentiation of myoblasts
Another factor which influences early myogenic differentiation positively is akirin-1 (also known as Mighty) (Salerno et al. 2009). In skeletal muscle tissue, akirin-1 is known to activate quiescent satellite cells and thus promote proliferation of muscle precursor cells. Furthermore, akirin-1 consecutively induces the expression of IGF-2 and hence also increases myogenic differentiation indirectly (Marshall et al. 2008). Therefore, akirin-1 combines the activation of quiescent satellite cells with the promyogenic effect of downstream growth factors such as IGFs. Though promising for skeletal muscle TE, the mechanism and molecular pathways of akirin-1 still have to be analysed in detail in future. In addition, the administration of growth factors has to be critically analyzed concerning their risk of tumorigenicity
Recently, a novel class of regulating factors of myogenesis has been analyzed for their promyogenic potential: Small non-coding RNAs, called microRNA (miRNA) which consist of approximately 20-22 nucleotides (Callis et al. 2008). Herein, certain microRNAs (miR-1, miR-133 and miR-206) have been demonstrated as muscle specific. Whereas miR-1 and miR-133 are also expressed in cardiac muscle, miR-206 is specifically expressed in skeletal muscle tissue and up-regulated in patients with muscular dystrophy (Eisenberg et al. 2009). Furthermore, the muscle-specific miRNAs differ in their effect on muscle precursor cells. MiR-133 increases proliferation of muscle precursor cells but also inhibits myogenic differentiation (Chen, J.F. et al. 2006). On the contrary, miR-1 and miR-206 have been shown to induce myogenic differentiation (Chen, J.F. et al. 2006, Kim, H.K. et al. 2006). In a recent study, Nakasa et al demonstrated that local injection of miR-1, miR-133 and miR-206 improves muscle regeneration and prevent fibrosis following muscle injury
5.2. Electrical stimulation and neurotization
Despite great efforts in the past and various molecular factors which regulate and enhance myogenesis, engineering of mature skeletal muscle tissue still remains a big challenge. Though contracting myotubes – which mark the differentiation and fusion of myoblasts in myogenesis - have been generated by various groups, the generation of adult muscle fibers depends on neural or electrical stimulation (Wilson & Harris 1993). The influence of electrical stimulation on further myogenic differentiation has been analyzed
However, engineering functional skeletal muscle tissue
The main component of the neuromuscular junction – the acetylcholine receptor (AChR) – is initially expressed in developing myofibers also in absence of neural cells, i.e. independent from motoric innervation (Witzemann 2006). The AChR clusters at this stage of myogenesis are located at the central regions of myofibers and this phenomenon known as “prepatterning”, marks the development of mature myofibers in myogenesis. However, the accumulation of AChR at synaptic sites and further development into functional neuromuscular junctions depends on the specific neuronal factor agrin. Thus, the molecular signalling between developing myofibers and motor neurons is necessary for the generation of functional neuromuscular junctions (Brockhausen et al. 2008). The motoric innervation even defines further maturation of the developing muscle tissue into slow- or fast-twitching muscle fibers (Nehrer-Tairych et al. 2000). Dhawan
As a conclusion, the main challenges in skeletal muscle TE are therefore: (1) engineering a suitable matrix for muscle TE including a clinical application, (2) improving further myogenic differentiation in vivo and (3) enabling the transplantation of functional skeletal muscle tissue to the recipient site including microsurgical anastomosis of an adequate vasculature as well as motoric neurotization of the engineered muscle tissue. Despite these obstacles, the achievements of the recent years demonstrate an encouraging progress of skeletal muscle TE research. Therefore, Churchill’s statement concerning skeletal muscle TE in vitro may still come true in the future.