**1. Introduction**

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Skeletal muscle injuries are common in humans, particularly in athletes and it is important to develop new methods to improve muscle regeneration. Skeletal muscle has good regener‐ ative ability, but the extent of muscle injury might prevent complete regeneration, especially in terms of functional recovery. Severe lesions, like those originated by trauma associated with loss of healthy muscular tissue and development of fibrous tissue scar and irreversible muscular atrophy after long-term peripheral nervous injuries are examples of those situa‐ tions where regeneration is limited. An alternative approach for the restoration of the dam‐ aged skeletal muscular tissue, considered to be an ultimate treatment of some traumatic or degenerative diseases, is the transplantation of stem cells that limit the fibrosis and the atro‐ phy of the involved muscle masses, and even imply the myocytes regeneration and local re‐ vascularization [1]. Stem cells and regenerative medicine is a fast emerging field with rapid strides of progress and focus on human health. Successful clinical use of stem cells in regen‐ erative medicine depends on 3 important features:


© 2013 Pereira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The purpose of this review is to describe the current research lines in the skeletal muscle re‐ generation field, with special emphasis to the work performed by our research group in test‐ ing different biomaterials and cellular therapies, emphasizing the use of mesenchymal stem cells (MSCs) isolated from the Wharton's jelly of the umbilical cord. We also focused our re‐ search in developing skeletal muscular lesion models which could be reproducible. It is im‐ portant to state, that a multidisciplinary team has a crucial role in the development of these biomaterials associated to cellular systems, and in pre-clinical tests. MSCs comprise a rare population of multipotent progenitor cells with a great therapeutic potential since they are capable of self-renewal and multi-lineage differentiation. Due to this ability, MSCs appear to be an attractive tool in the context of Tissue Engineering and cell-based therapy concerning skeletal muscle regeneration. Several biomaterials associated to MSCs from the Wharton's jelly of the umbilical cord have been tested in standard lesions of the rat muscle and the re‐ sults of these tests will be discussed here. The umbilical cord matrix is an important and safe source of MSCs with positive effects in nerve and skeletal muscle regeneration, with no ethi‐ cal or technical issues.

#### **2. Skeletal muscle tissue**

#### **2.1. Basic structure and terminology**

The muscle fibers are the basic contractile units of skeletal muscles. They are individually surrounded by a connective tissue layer and grouped into bundles to form a skeletal muscle [1]. Each muscle is surrounded by a layer of dense connective tissue - the epimysium which is continuous with the tendon. The muscle is composed of numerous bundles of mus‐ cle fibers – fascicles - which are separated from each other by another connective tissue layer named perimysium. The endomysium is the connective tissue that separates individual muscle fibers from each other. Mature muscle cells are termed muscle fibers or myofibers. Each myofiber is a multinucleate syncytium formed by fusion of immature muscle cells termed myoblasts. In the cytoplasm of each myofiber – the sarcoplasm - lays the contractile apparatus of the cell which is composed of sarcomeres arranged in series to form myofibrils, which give myofibers their striated appearance. The sarcomeres contain a number of pro‐ teins, including alpha-actinin — which is the major constituent of the Z band — and actin and myosin, which are the major components of the thin and thick filaments, respectively. The sarcoplasm, located between the myofibrils, is called the intermyofibrillar network and contains the mitochondria, lipid, glycogen, T-tubules, and sarcoplasmic reticulum [1-3]. Skeletal muscles are highly vascularized to provide essential nutrients for muscle function. As the myofiber matures, it is contacted by a single motoneuron and expresses characteristic molecules for contractile function, principally different myosin heavy chain isoforms and metabolic enzymes. Both the motoneuron and the myoblast origin have been implicated to play a role in specifying the myofiber contractile properties, although the precise mecha‐ nisms remain to be defined [1].

#### *2.1.1. Fiber type*

The purpose of this review is to describe the current research lines in the skeletal muscle re‐ generation field, with special emphasis to the work performed by our research group in test‐ ing different biomaterials and cellular therapies, emphasizing the use of mesenchymal stem cells (MSCs) isolated from the Wharton's jelly of the umbilical cord. We also focused our re‐ search in developing skeletal muscular lesion models which could be reproducible. It is im‐ portant to state, that a multidisciplinary team has a crucial role in the development of these biomaterials associated to cellular systems, and in pre-clinical tests. MSCs comprise a rare population of multipotent progenitor cells with a great therapeutic potential since they are capable of self-renewal and multi-lineage differentiation. Due to this ability, MSCs appear to be an attractive tool in the context of Tissue Engineering and cell-based therapy concerning skeletal muscle regeneration. Several biomaterials associated to MSCs from the Wharton's jelly of the umbilical cord have been tested in standard lesions of the rat muscle and the re‐ sults of these tests will be discussed here. The umbilical cord matrix is an important and safe source of MSCs with positive effects in nerve and skeletal muscle regeneration, with no ethi‐

The muscle fibers are the basic contractile units of skeletal muscles. They are individually surrounded by a connective tissue layer and grouped into bundles to form a skeletal muscle [1]. Each muscle is surrounded by a layer of dense connective tissue - the epimysium which is continuous with the tendon. The muscle is composed of numerous bundles of mus‐ cle fibers – fascicles - which are separated from each other by another connective tissue layer named perimysium. The endomysium is the connective tissue that separates individual muscle fibers from each other. Mature muscle cells are termed muscle fibers or myofibers. Each myofiber is a multinucleate syncytium formed by fusion of immature muscle cells termed myoblasts. In the cytoplasm of each myofiber – the sarcoplasm - lays the contractile apparatus of the cell which is composed of sarcomeres arranged in series to form myofibrils, which give myofibers their striated appearance. The sarcomeres contain a number of pro‐ teins, including alpha-actinin — which is the major constituent of the Z band — and actin and myosin, which are the major components of the thin and thick filaments, respectively. The sarcoplasm, located between the myofibrils, is called the intermyofibrillar network and contains the mitochondria, lipid, glycogen, T-tubules, and sarcoplasmic reticulum [1-3]. Skeletal muscles are highly vascularized to provide essential nutrients for muscle function. As the myofiber matures, it is contacted by a single motoneuron and expresses characteristic molecules for contractile function, principally different myosin heavy chain isoforms and metabolic enzymes. Both the motoneuron and the myoblast origin have been implicated to play a role in specifying the myofiber contractile properties, although the precise mecha‐

cal or technical issues.

**2. Skeletal muscle tissue**

nisms remain to be defined [1].

**2.1. Basic structure and terminology**

330 Advances in Biomaterials Science and Biomedical Applications

Individual adult skeletal muscles are composed of a mixture of myofibers with different physiological properties, ranging from a slow-contracting/fatigue-resistant type to a fastcontracting/non-fatigue-resistant type. The proportion of each fiber type within a muscle de‐ termines its overall contractile properties [1]. The slow contracting *soleus* muscle is rich in myofibers expressing the slow type I myosin heavy chain isoform, whereas the fast contract‐ ing plantaris muscle is devoid of slow type I myofibers [1-3]. The most informative methods to delineate muscle fiber types are based on specific myosin profiles, specially the myosin heavy chain (MHC) isoform complement. According to the major MHC isoforms found in adult mammalian skeletal muscles, the following pure fiber types exist: slow type I with MHCIb, and three fast types, namely type IIA with MHCIIa, type IID with MHCIId and type IIB with MHCIIb [4]. Despite having different physiological properties, the basic mech‐ anism of muscle contraction is similar in all myofiber types and is the result of a "sliding mechanism" of the myosin-rich thick filament over the actin-rich thin filament after neuro‐ nal activation [5]. The connective tissue framework in skeletal muscle combines the contrac‐ tile myofibers into a functional unit, in which the contraction of myofibers is transformed into movement via myotendinous junctions at their ends, where myofibers attach to the skeleton by tendons. Thus the functional properties of skeletal muscle depend on the main‐ tenance of a complex framework of myofibers, motor neurons, blood vessels, and extracellu‐ lar connective tissue matrix [1].

#### **2.2. Regeneration of the skeletal muscle**

Regeneration is a unique adaptation of skeletal muscle that occurs in response to injury. Following direct trauma or disease, the regeneration of skeletal muscle results in restora‐ tion, to some degree, of the original structure and function of the muscle tissue [6]. Skeletal muscle regeneration is a physiological response of the tissue to traumatic or pathological injuries and its progress depends on the type of damaged muscle and the extent of the injury. Under normal conditions, the regenerated muscle is morphologically and functionally indis‐ tinguishable from undamaged muscle [1]. Regeneration resembles the process of formation of skeletal muscle during embryogenesis. Skeletal myogenesis begins in the somites where multipotencial mesodermal cells commit to the myogenic lineage. These mononucleated my‐ oblasts then fuse and form multinucleated cells (myotubes) that ultimately develop into mature myofibers [1, 7]. During the course of muscle development, a distinct subpopulation of myo‐ blasts fails to differentiate and remains associated with the surface of the developing myofib‐ er as quiescent muscle satellite cells (SCs) in fully developed mature skeletal tissue [1, 7].

#### *2.2.1. Satellite Cells and other cells involved in regeneration of skeletal muscle tissue*

During regeneration and muscle repair, SCs fuse together or to the existing fibers to form new muscle fibers [8]. Although the number of SCs is greatly reduced in aged muscle, those remaining maintain an intrinsic capacity to regenerate the muscle tissue as efficiently as in younger muscles. A vital condition for successful regeneration is the presence of SCs in the uninjured portions of the basal membrane of the myofiber, along with its ability for reinner‐ vation and revascularization. After a skeletal muscle injury, myofibers become completely desintegrated via myolysis and the SCs are realeased from the basal membrane. From this point SCs start to divide and are capable of differentiating into muscle fibers, reestablishing myofiber's architecture and restoring the muscle function [9, 10]. In post-natal skeletal mus‐ cle, PW1 expression is detected in SCs and a subset of interstitial cells and is markedly upregulated during muscle regeneration [11]. These interstitial multipotent stem cells are extralaminal and exhibit fibroblastic morphology but do not express the same myogenic markers such as Pax7 [10]. PW1+ /Pax7– interstitial cells (PICs) are myogenic *in vitro* and effi‐ ciently contribute to skeletal muscle regeneration *in vivo* as well as generating satellite cells and PICs. PICs show bipotential behavior *in vitro,* generating both smooth and skeletal mus‐ cle. Isolated PICs do not express Pax7 or MyoD, but they convert to a Pax7+ /MyoD+ state be‐ fore forming skeletal muscle *in vitro.* PICs are not derived from a Pax3-expressing parental cell and thus do not share a satellite cell lineage; however, PICs do express Pax3 upon con‐ version to skeletal muscle. PICs are a key cell population that cannot be recruited into the skeletal muscle lineage in the absence of Pax7 function and is likely to contribute to the Pax7 muscle phenotype during postnatal growth. PICs are as abundant as SCs in muscle tissue and correspond to the only population of PW1+/Pax7– cells *in vivo*, requiring Pax7 for their myogenic capacity [11]. PDGFRα+ mesenchymal progenitor cells located in the muscle *inter‐ stitium* were also identified as being distinct from SCs. Of the muscle-derived cell popula‐ tions, only PDGFRα+ cells show efficient adipogenic differentiation both *in vitro* and *in vivo*, being strongly inhibited by the presence of satellite cell-derived myofibres. These results suggest that PDGFRα+ mesenchymal progenitors are the major contributor to ectopic fat cell formation in skeletal muscle that is more conspicuous in perimysium and particularly in perivascular space. The balance between satellite cell-dependent myogenesis and PDGFRα+ cell-dependent adipogenesis, rather than multipotency of satellite cells, has a considerable impact on muscle homeostasis [12]. Hematopoietic and dendritic cells are also present in the perimysium of the skeletal tissue, as well as some lymphocytes and macrophages [10].

#### *2.2.2. Myogenic differentiation*

Cells derived from Pax3-expressing cells are myofibres and SCs [11]. Once activated, SCs ex‐ press factors involved in the specification of the myogenic program, such as Pax-7, desmin, MNFα, Myf5, MRF4 and MyoD. Activated SCs enter the cell cycle and proliferate as indicat‐ ed by the expression of factors involved in cell cycle progression, such as PCNA and by the incorporation of BrDU. Recently, miRNAs have also been reported to regulate gene expres‐ sion in skeletal muscle. Upon activation, SCs generate fusion-competent myoblasts and can self-renew at least to a limited extent. Any interruption in the proliferation or fusion of myo‐ blasts, or any alterations in the extracellular matrix leads to the development of fibrosis, compromising the establishment of the correct muscular function [8, 10]. Proliferative MyoD and/or Myf5 positive myogenic cells are termed myoblasts. Both SCs and myoblasts increase their cytoplasmic-nuclear ratio and can migrate along myofibers. Proliferating myoblasts withdraw from the cell cycle to become terminally differentiated myocytes that express the "late" myogenic regulatory factors (MRFs), Myogenin and MRF4, and subsequently musclespecific genes such as MHC and muscle creatine kinase (CKM), and stopping Pax7 expres‐ sion. Myogenic subpopulations have also been identified by their enriched M-cadherin and CD34 expression. M-cadherin can be considered to be a reliable marker for both quiescent and activated SCs. Once fusion of myogenic cells is completed, newly formed myofibers in‐ crease in size and myonuclei move to the periphery of the muscle fiber [1, 10, 13].

#### *2.2.3. Degeneration*

vation and revascularization. After a skeletal muscle injury, myofibers become completely desintegrated via myolysis and the SCs are realeased from the basal membrane. From this point SCs start to divide and are capable of differentiating into muscle fibers, reestablishing myofiber's architecture and restoring the muscle function [9, 10]. In post-natal skeletal mus‐ cle, PW1 expression is detected in SCs and a subset of interstitial cells and is markedly upregulated during muscle regeneration [11]. These interstitial multipotent stem cells are extralaminal and exhibit fibroblastic morphology but do not express the same myogenic

ciently contribute to skeletal muscle regeneration *in vivo* as well as generating satellite cells and PICs. PICs show bipotential behavior *in vitro,* generating both smooth and skeletal mus‐

fore forming skeletal muscle *in vitro.* PICs are not derived from a Pax3-expressing parental cell and thus do not share a satellite cell lineage; however, PICs do express Pax3 upon con‐ version to skeletal muscle. PICs are a key cell population that cannot be recruited into the skeletal muscle lineage in the absence of Pax7 function and is likely to contribute to the Pax7 muscle phenotype during postnatal growth. PICs are as abundant as SCs in muscle tissue and correspond to the only population of PW1+/Pax7– cells *in vivo*, requiring Pax7 for their myogenic capacity [11]. PDGFRα+ mesenchymal progenitor cells located in the muscle *inter‐ stitium* were also identified as being distinct from SCs. Of the muscle-derived cell popula‐ tions, only PDGFRα+ cells show efficient adipogenic differentiation both *in vitro* and *in vivo*, being strongly inhibited by the presence of satellite cell-derived myofibres. These results suggest that PDGFRα+ mesenchymal progenitors are the major contributor to ectopic fat cell formation in skeletal muscle that is more conspicuous in perimysium and particularly in perivascular space. The balance between satellite cell-dependent myogenesis and PDGFRα+ cell-dependent adipogenesis, rather than multipotency of satellite cells, has a considerable impact on muscle homeostasis [12]. Hematopoietic and dendritic cells are also present in the perimysium of the skeletal tissue, as well as some lymphocytes and macrophages [10].

Cells derived from Pax3-expressing cells are myofibres and SCs [11]. Once activated, SCs ex‐ press factors involved in the specification of the myogenic program, such as Pax-7, desmin, MNFα, Myf5, MRF4 and MyoD. Activated SCs enter the cell cycle and proliferate as indicat‐ ed by the expression of factors involved in cell cycle progression, such as PCNA and by the incorporation of BrDU. Recently, miRNAs have also been reported to regulate gene expres‐ sion in skeletal muscle. Upon activation, SCs generate fusion-competent myoblasts and can self-renew at least to a limited extent. Any interruption in the proliferation or fusion of myo‐ blasts, or any alterations in the extracellular matrix leads to the development of fibrosis, compromising the establishment of the correct muscular function [8, 10]. Proliferative MyoD and/or Myf5 positive myogenic cells are termed myoblasts. Both SCs and myoblasts increase their cytoplasmic-nuclear ratio and can migrate along myofibers. Proliferating myoblasts withdraw from the cell cycle to become terminally differentiated myocytes that express the "late" myogenic regulatory factors (MRFs), Myogenin and MRF4, and subsequently musclespecific genes such as MHC and muscle creatine kinase (CKM), and stopping Pax7 expres‐

interstitial cells (PICs) are myogenic *in vitro* and effi‐

/MyoD+

state be‐

/Pax7–

cle. Isolated PICs do not express Pax7 or MyoD, but they convert to a Pax7+

markers such as Pax7 [10]. PW1+

332 Advances in Biomaterials Science and Biomedical Applications

*2.2.2. Myogenic differentiation*

This scenario changes dramatically when the muscle is damaged, in which muscle degener‐ ation after acute injury is characterized by myofiber necrosis and is followed by inflamma‐ tion, tissue reconstruction and remodeling [10]. The necrosis is triggered by disruption of the myofiber sarcolemma resulting in increased myofiber permeability. The disruption of myofiber integrity is reflected by increased serum levels of muscle proteins, such as CK (usually restricted to the myofiber cytosol) [1]. It has been hypothesized that increased Ca2+ influx after sarcolemmal or sarcoplasmic reticulum damage results in a loss of Ca2+ homeo‐ stasis and increased Ca2+-dependent proteolysis that drives tissue degeneration resulting in focal or total autolysis depending on the extent of the injury [1].

#### *2.2.4. Inflammation*

The early regenerative response in skeletal muscle is similar to that in other tissues and requires the coordinated regulation of inflammation, extracellular matrix remodeling, and myofiber growth [14]. The early phase of muscle injury is usually accompanied by the activation of mononucleated cells, mainly inflammatory cells and myogenic cells. Factors released by the injured muscle activate inflammatory cells residing within the muscle, which in turn pro‐ vide the chemotactic signals to circulating immune cells. Neutrophils are the first immune cells to invade the injured muscle, with a significant increase in their number being ob‐ served as early as 1–6h after myotoxin or exercise-induced muscle damage. After neutrophil infiltration and 48h post-injury, macrophages become the predominant inflammatory cell type within the site of injury. Macrophages infiltrate the injured site and through phagocytosis remove cellular debris and may affect other aspects of muscle regeneration by activating myogenic cells [1]. Testosterone has a documented ability to modulate the activity of im‐ mune, fibroblast, and myogenic precursor cells, which are all components of regeneration [14].
