**4. Biomimetic models of muscle**

**Cells** - Skeletal muscle cells can be cultured *in vitro* following their isolation from muscle tissue. The main advantage of *in vitro* cultures of skeletal muscle precursor cells (MPCs) is the faithful recapitulation of the events that occur *in vivo* during development and regeneration whereby mononuclear MPCs fuse together under the correct conditions to form multinucleate myotubes - essentially nascent muscle fibres. Skeletal muscle development occurs in the somites; segments of paraxial mesoderm which form on either side of the neural tube [10], which is further divided into the epaxial and hypaxial dermomyotome [11]. Cells in the dermomyotome migrate and form the myotome, and transiently express the paired box transcription factors Pax 3 and 7, and Muscle Regulatory Factors (MRFs) Myf5, MyoD and Myogenin [12], which regulate myotube formation. The expression of these transcription factors has been shown to be critical for 'normal' muscle formation during development [13, 14] and null mutations of some of these genes can be lethal during the early post-natal period [15, 16]. Whilst the myotome gives rise to the musculature of the trunk, Pax 3+ cells migrate out of the hypaxial

<sup>3</sup> http://www.defra.gov.uk/

<sup>4</sup> http://www.nerc.ac.uk/

<sup>5</sup> http://erc.europa.eu/

<sup>6</sup> http://www.wellcome.ac.uk/

dermomyotome to the limb bud where they form the musculature of the limbs [10] under the influence of MRFs.

research. Developed in consultation with the scientific community, including researchers, statisticians, journal editors, and funders, the guidelines request essential information that should be included in publications reporting animal research. Essentially the guidelines are intended to improve reporting of research using animals; guide authors as to the essential information to include in a manuscript, and not be absolutely prescriptive; be flexible to accommodate reporting a wide range of research areas and experimental protocols; promote reproducible, transparent, accurate, comprehensive, concise, logically ordered, well written manuscripts and improve the communication of the research findings to the broader scientific community. The UK's major bioscience funding bodies, including the Medical Research Council, the Biotechnology and Biological Sciences Research Council, Department for Envi‐

rated adherence to the guidelines into a revised version of 'Responsibility in the use of animals in bioscience research: Expectations of the major research council and charitable funding

Although the ARRIVE guidelines are primarily aimed at scientists writing up their research for publication and for those who are involved in peer review and intended to improve standards of reporting data from animal experiments, they have also highlighted the potential of biomimetic and tissue engineered *in vitro* models for replacing, refining and reducing the

**Cells** - Skeletal muscle cells can be cultured *in vitro* following their isolation from muscle tissue. The main advantage of *in vitro* cultures of skeletal muscle precursor cells (MPCs) is the faithful recapitulation of the events that occur *in vivo* during development and regeneration whereby mononuclear MPCs fuse together under the correct conditions to form multinucleate myotubes - essentially nascent muscle fibres. Skeletal muscle development occurs in the somites; segments of paraxial mesoderm which form on either side of the neural tube [10], which is further divided into the epaxial and hypaxial dermomyotome [11]. Cells in the dermomyotome migrate and form the myotome, and transiently express the paired box transcription factors Pax 3 and 7, and Muscle Regulatory Factors (MRFs) Myf5, MyoD and Myogenin [12], which regulate myotube formation. The expression of these transcription factors has been shown to be critical for 'normal' muscle formation during development [13, 14] and null mutations of some of these genes can be lethal during the early post-natal period [15, 16]. Whilst the myotome gives rise to the musculature of the trunk, Pax 3+ cells migrate out of the hypaxial

), the Natural Environment Research Council

, have all incorpo‐

) and the Wellcome Trust6

ronment, Food and Rural Affairs (Defra3

512 Regenerative Medicine and Tissue Engineering

use of laboratory animals in research.

**4. Biomimetic models of muscle**

3 http://www.defra.gov.uk/ 4 http://www.nerc.ac.uk/ 5 http://erc.europa.eu/ 6 http://www.wellcome.ac.uk/

), the European Research Council (ERC5

bodies', which was originally published in May 2008.

(NERC4

A subset of Pax 7/3+ cells have been identified in the limb buds and myotome in development, but fail to differentiate to form myotubes [17]. These proliferating precursor cells can adopt a position between the developed myofibre and the basal lamina during later muscle develop‐ ment [17, 18], which strongly indicates that this subset of progenitors form the satellite cells of post-natal skeletal muscle.

Satellite cells are the resident stem cells of skeletal muscle tissue, which provide additional nuclei to a muscle fibre during regeneration, such as may occur following muscle injury, damage or overload. Myonuclear addition is important in order for the muscle to effectively synthesise new proteins and thus adapt to specific stimuli, as well as generally maintain and increase its mass. Satellite cells are so called due to their anatomical location between the sarcolemma and basal lamina of a muscle fibre [19], and this parameter was for some time the only true 'marker' of these cells, achievable via electron microscopy. More recently however, a host of proteins which are expressed by satellite cells have been identified, including Pax 7 [20], Caveolin-1 [21] and myf-5 [22], and thus a molecular signature of these cells is being derived.

Satellite cells lie quiescent on the muscle periphery until becoming activated upon muscle damage/overload. The precise cause of satellite cell activation remain elusive7 . However the fact that muscle injury leads to activation suggests that muscle damage itself may be the trigger or a factor secreted from the muscle may provide the initial cue [23]. The latter notion has support from the fact that Insulin Like Growth Factor-I (IGF-I) and indeed Hepatocyte Growth Factor (HGF) are released from the muscle following mechanical use or injury [24-29] and have been shown to activate satellite cells when exogenously administered [30-35]. Once activated, satellite cells proliferate extensively and express the transcription factors myf-5 and MyoD [36] as well as proteins specific to the myogenic lineage such as Desmin, before fusing with the damaged/overloaded fibres under the influence of myogenin [37] to provide additional myonuclei. As stem cells, satellite cells have the ability to self-renew and replenish the pool of quiescent cells following activation. The literature suggests that asymmetric distribution of a number of potential proteins is implicated in regulating self-renewal [38-40], causing some daughter cells to express pax 7 and MyoD, and thus commit to the myogenic lineage and undergo fusion, whereas a small portion fail to express MyoD or myf-5 and thus repopulate the satellite cell pool [38, 39, 41].

Isolation and subsequent culture of MPCs (satellite cells when in their anatomical niche) can be conducted either by explant culture, whereby muscle tissue is minced and maintained in culture until MPCs migrate from the tissue [42, 43], or via enzymatic digestion, whereby the tissue is broken down completely to release all of the resident mononuclear cells [44, 45]. Following isolation, careful consideration of the culture techniques should be taken in order to standardise/optimise the desired experiments. Indeed, MPCs are sensitive to the media

<sup>7</sup> Neil R.W. Martin and Mark P. Lewis.Satellite cell activation and number following acute and chronic exercise: A mini review. Cellular and Molecular Exercise Physiology 1[1]: e3. doi:10.7457/cmep.v1i1.e3

composition, and will either proliferate or exit the cell cycle and differentiate when exposed to various media compositions [46]. Furthermore, MPCs are sensitive to environmental factors, and will proliferate and fail to differentiate when exposed to low oxygen levels [47, 48] and similarly, serial passaging of MPCs also appears to negatively effect their ability to differentiate [49]. Finally, as the population of cells released from skeletal muscle tissue tends to be mixed in nature (e.g. MPCs, fibroblasts, pericytes, endothelial cells etc.), it is often favourable to further purify the MPCs prior to experimentation either via differential adhesion methods or magnetic separation, a methodology optimise in our laboratory [50], however these cells may contribute to the development of more biomimetic tissue (detailed in Figure 1 below).

**Figure 1.** Skeletal muscle precursor cells can be isolated and cultured *in vitro* to recapitulate the events of develop‐ ment and regeneration. (A). Phase contrast photomicrograph of human skeletal muscle explant with MPCs migrating away from the tissue. (B) Immunostaining for Desmin (green) highlights those cells, which are commited to the myo‐ genic lineage. (C) Desmin positive cells form multinucleate myotubes when stimulated to do so. Scale bars = 100µm.

The isolation and characterisation of cells for utilisation in the development of *in vitro* skeletal muscle models is an important stage in the engineering process. However, the consideration of the influence of environmental factors in the creation of a biomimetic model is a necessity and will be explored below.

**Environment** - The skeletal muscle cell microenvironment (commonly referred to as the niche) supports the development of multi-nucleate myotubes (muscle fibres) in a number of ways: provision of directional cues, provision of topographical cues, availability of growth factors, transmission of mechanical signals and interactions with other cell types. Initial evidence that particular proteins within the skeletal muscle cell niche play critical roles in these processes and hence subsequent success of *in vitro* muscle cell behaviour, came with the establishment of the varying effects of ECM molecules on MPC proliferation and differentiation. Extracting bioactive ECM proteins from skeletal muscle for use as a growth substrate with *in vitro* MPC culture, results in superior proliferation and differentiation compared to standard growth substrates [51]. It is however the identification of interactions between ECM proteins and muscle cell surface proteins that has provided data regarding the requirements for successful *in vitro* culture of MPCs.

composition, and will either proliferate or exit the cell cycle and differentiate when exposed to various media compositions [46]. Furthermore, MPCs are sensitive to environmental factors, and will proliferate and fail to differentiate when exposed to low oxygen levels [47, 48] and similarly, serial passaging of MPCs also appears to negatively effect their ability to differentiate [49]. Finally, as the population of cells released from skeletal muscle tissue tends to be mixed in nature (e.g. MPCs, fibroblasts, pericytes, endothelial cells etc.), it is often favourable to further purify the MPCs prior to experimentation either via differential adhesion methods or magnetic separation, a methodology optimise in our laboratory [50], however these cells may contribute to the development of more biomimetic tissue (detailed in Figure 1 below).

**Figure 1.** Skeletal muscle precursor cells can be isolated and cultured *in vitro* to recapitulate the events of develop‐ ment and regeneration. (A). Phase contrast photomicrograph of human skeletal muscle explant with MPCs migrating away from the tissue. (B) Immunostaining for Desmin (green) highlights those cells, which are commited to the myo‐ genic lineage. (C) Desmin positive cells form multinucleate myotubes when stimulated to do so. Scale bars = 100µm.

The isolation and characterisation of cells for utilisation in the development of *in vitro* skeletal muscle models is an important stage in the engineering process. However, the consideration of the influence of environmental factors in the creation of a biomimetic model is a necessity

**Environment** - The skeletal muscle cell microenvironment (commonly referred to as the niche) supports the development of multi-nucleate myotubes (muscle fibres) in a number of ways: provision of directional cues, provision of topographical cues, availability of growth factors, transmission of mechanical signals and interactions with other cell types. Initial evidence that particular proteins within the skeletal muscle cell niche play critical roles in these processes and hence subsequent success of *in vitro* muscle cell behaviour, came with the establishment of the varying effects of ECM molecules on MPC proliferation and differentiation. Extracting bioactive ECM proteins from skeletal muscle for use as a growth substrate with *in vitro* MPC culture, results in superior proliferation and differentiation compared to standard growth substrates [51]. It is however the identification of interactions between ECM proteins and

and will be explored below.

514 Regenerative Medicine and Tissue Engineering

Particularly, integrins and proteins of the Dystrophin Associated Protein (DAP) complex have been demonstrated to play significant roles in MPC-ECM interaction *in vitro*. Indeed, the expression of α v, α v β 3 and α v β 5 integrins is evident in MPCs seeded on ECM substrates such as gelatin, fibronectin and vitronectin [52]. Moreover, blockade of α v integrin using a specific antibody abolishes cell migratory capacity on fibronectin and vitronectin substrates [52]. The effect of ECM matrix components has also been demonstrated to influence the contractile phenotype of engineered skeletal muscle constructs. Increasing the content of ECM protein rich Matrigel, contributed to greater peak force of engineered constructs, whilst utilising growth factor reduced Matrigel did not affect the results [53]. This suggests that ECM matrix composition contributes to skeletal muscle maturation and phenotype to a greater extent than growth factors. These data provide clear evidence as to the importance of the ECM in the successful culture of MPCs *in vitro*.

The primary function of skeletal muscle *in vivo* is to provide vector force, for the controlled movement of the skeleton [54]. This directionality is achieved by the highly organised macro and microstructure of the skeletal muscle fibres and the surrounding ECM8 . The recapitulation of this organised structure has been achieved through a variety of methodologies *in vitro*, including the use of both synthetic [55-58] and naturally derived polymers [45, 59-63]. These methods have provided both directional and topographical cellular cues, display varying amounts of resemblance and functionality to *in vivo* tissue. It has been suggested however, that the rigidity of synthetic polymers may affect the contractile properties and function of developed myotubes, whilst if the polymer is bio-degradable the alignment of cells may be affected by this process [64]. The use of naturally derived polymers (including fibrin and collagen) has in part been shown to overcome these issues. Particularly in a more biomimetic three-dimensional (3-D) structure, the rapid polymerisation of collagen matrices allows for an even distribution of the cells seeded, whilst the nanostructure allows for multiple attachment sites for the cells [64].

TheprovisionofmechanicalsignalsforthealignmentandfusionofMPCsisanessentialcriterion for developing biomimetic constructs *in vitro*, since skeletal muscle *in vivo* is under continuous passive strain through the myotendinous junction. Such signals have been generated by the polymerisationof a biological substrate to a fixedstructure ina variety of*in vitro* skeletalmuscle models [45, 53, 65, 66]. The anchored structures provide the necessary passive lines of strain, which provide the required signals to generate an orientated cellular architecture (See Figure 2 below). Indeed, it is clear that the 3-D structure and the presence of passive mechanical signals promote the alignment and fusion of MPCs to a greater extent than in conventional monolayer culture [45]. Furthermore, the morphological structure, gene expression and contractile phenotype are also representative of *in vivo* skeletal muscle [43, 45, 67].

<sup>8</sup> Alec S.T. Smith, Rishma Shah, Nigel P. Hunt, Mark P. Lewis. The Role of Connective Tissue and Extracellular Matrix Signaling in Controlling Muscle Development, Function, and Response to Mechanical Forces. Seminars in orthodontics 1 June 2010 (volume 16 issue 2 Pages 135-142 DOI: 10.1053/j.sodo.2010.02.005)

**Figure 2.** Biomimetic skeletal muscle constructs can be engineered from biological scaffolds. Example model of the typical macroscopic contraction of a type-1 collagen matrix, following 14 days in culture. Note the custom made an‐ chors at either end, which provide predictable lines of longitudinal tension (illustrated by dashed arrows) to promote cell alignment and fusion in a single plane. Scale bar = 10 mm.

A further advantage of using naturally derived polymers is the ability to easily stimulate, both mechanically and electrically, due to the innate mechanical compliance of the polymerised tissue. To this end, numerous investigations have employed such techniques to promote myogenesis and maturation of seeded MPCs [65, 68, 69]. In contrast, the effect of a combined mechanical stimulation protocol, contributed to a reduction in maturation of 3-D seeded myogenic cells compared to monolayer controls [66]. Nevertheless, the effect of increased mechanical signals for the promotion of MPC alignment and fusion, satisfies the desire for a biomimetic.

Skeletal muscle *in vivo* is comprised of myogenic progenitors, non-myogeninc progenitors and fibroblasts. The presence and influence of these non-myogenic populations has not been widely investigated, however, there is evidence to suggest a potential synergistic role in the development and maturation of skeletal muscle *in vitro*. The non-myogenic fibroblast popu‐ lation of cells has been shown to enhance skeletal muscle cell alignment, through contact dependent and independent mechanisms [70]. Data from 3-D skeletal muscle constructs also demonstrates an increased myogenin mRNA expression (marker of terminal differentiation) and increased peak force in a heterogeneous mixture of myogenic and non-myogenic cells, compared to myogenic only cultures [43]. Furthermore the expression of MMP-2 mRNA was greater in the heterogeneous cell population cultures, suggestive of greater matrix remodelling and fusion potential [43]. Together, these data illustrate the requirement of the non-myogenic cell population to recapitulate the *in vivo* skeletal muscle cell niche and associated biological processes. As such the incorporation of a heterogeneous population of cells should be utilised when developing skeletal muscle models *in vitro*.

Consideration of the body of data discussed above, indicates that an argument can be made that the ideal solution for a tissue engineered skeletal muscle construct would embrace the following:


Provision of all of the cues simultaneously requires an approach that moves beyond conven‐ tional monolayer models. The structure defines function in skeletal muscle *in vivo* and all of the essential elements above can be combined using tissue engineering techniques. There are a number of systems that have moved towards this aim, however important validations for such systems is making comparisons against the *in vivo* tissue, physiological skeletal muscle. The systems described have shown the following features to date:


**Table 1.** Comparison of current biomimetic skeletal muscle models of *in vivo* skeletal muscle tissue.

There can now be a high degree of confidence that the aforementioned systems do recreate a significant aspect of *in vivo* skeletal muscle biology. Their use to-date with respect to providing a pre-clinical test bed for exercise, genetic, pharmaceutical and nutritional therapies is limited, however the system are now at a stage where such investigations are warranted.
