Engineered Matrices for Skeletal Muscle Satellite Cell Engraftment and Function

Abstract

Regeneration of traumatically injured skeletal muscles is severely limited. Moreover, the regenerative capacity of skeletal muscle declines with aging, further exacerbating the problem. Recent evidence support that delivery of muscle satellite cells to the injured muscles enhances muscle regeneration and reverses features of aging, including reduction in muscle mass and regenerative capacity. However, direct delivery of satellite cells presents a challenge at a translational level due to inflammation and donor cell death, motivating the need to develop engineered matrices for muscle satellite cell delivery. This review will highlight important aspects of satellite cell and their niche biology in the context of muscle regeneration, and examine recent progresses in the development of engineered cell delivery matrices designed for skeletal muscle regeneration. Understanding the interactions of muscle satellite cells and their niche in both native and engineered systems is crucial to developing muscle pathology-specific cell- and biomaterial-based therapies.

Introduction

Skeletal muscles function to generate force, enable locomotion, support posture, regulate metabolism, and secrete myokines with autocrine, paracrine and endocrine effects. Muscle has the capacity to repair itself upon minor strains, contusions, and lacerations; however, its native regenerative capacity with an onset of traumatic injury is severely restricted, often leading to further loss of muscle mass, fibrosis, and diminished function [1–3]. Furthermore, this problem is exacerbated in elderly patients, as muscle regenerative capacity declines with aging [4–7]. To date, effective clinical strategies to restore the native muscle tissue structure and function are extremely limited. Collectively, these problems pose significant socioeconomic costs and impact the quality of life [1,2,8,9]. For instance, in the United States, the estimated direct healthcare costs associated with sarcopenia alone exceeded $18.5 billion [8] and combined direct and indirect (i.e., lost productivity) costs associated with traumatic injury exceeded $406 billion in 2000 [10]. Despite the lack of more recent and reliable healthcare cost estimates of sarcopenia and its co-morbidities, the socioeconomic burden is projected to be even more significant with increasing number of individuals aged 65 years and older [9].

Recent advances in the stem cell and niche biology of muscle have identified muscle satellite cells (MuSCs) as one of the crucial components in muscle development and endogenous reparative processes [11,12]. MuSCs, which constitute approximately 2–10% of the total number of myonuclei [13], are quiescent in healthy and resting muscles, but are activated for proliferation and differentiation to form new myofibers in response to mechanical and chemical stimuli resulting from injurious events [11,12]. Indeed, delivery of MuSCs into the injured intramuscular space enhances regenerative outcomes [14–16], indicating that stem cell therapy may be a promising strategy for treating muscle injuries and disorders. Furthermore, it is also important to highlight the role of the MuSC niche in the regenerative process. In particular, recent evidence suggests that biochemical factors (e.g., growth factors) from both systemic and local milieu, as well as biophysical factors (e.g., matrix stiffness, adhesive ligands) significantly influence the bioactivity of the MuSCs and their functional coordination with other resident cells (e.g., inflammatory and fibroadipogenic progenitor cells) [11,17–20], ultimately impacting regenerative outcomes. The intricate interactions of MuSCs and their niche during homeostasis and regeneration are also important considerations in the context of aging, where age-associated alterations in the MuSC niche will negatively influence the function of the skeletal muscle, and further contribute to the progression of sarcopenia [7].

Delivery of stem cells is an effective approach to stimulate and enhance skeletal muscle regeneration in vivo [14–16]. However, direct intramuscular delivery of MuSCs presents challenges at the translational level, as the transplanted MuSCs exhibit limited migration in vivo and are exposed to inflammatory microenvironment consisting of neutrophils, macrophages, reactive oxygen species, and pro-inflammatory cytokines upon injury, where such signals induce cell death and inadequate cellular function [22]. In order to maintain the viability and enhance functional outcomes, engineered biomaterials designed to transiently protect the donor MuSCs from the inflammatory environment and to direct their function in vivo are needed. Specifically, elucidating how MuSCs interact with their niche, as well as incorporating key elements of the niche in the engineered delivery matrices are crucial to developing the optimal bio-functional delivery matrix for skeletal muscle regeneration. Further challenges in cell- and biomaterial-based therapies in the context of skeletal muscle regeneration include having precise control over immune-modulation, as well as effective stimulation of innervation and vascularization. For instance, MuSCs play an important role in forming neuromuscular junctions [23], where quality of neuromuscular junction and motoneuron innervation is important for normal muscle function and susceptibility sarcopenia in rodents and human [24]. This suggests that modulating innervation of the transplanted MuSCs is an important consideration for cell- and biomaterial-based therapy to improve regenerative outcomes in both acute injury and aging contexts. Such considerations to promote vascularization and innervation have been previously reviewed in greater detail [25].

This review will first highlight recent advances in the understanding of MuSCs and their niche in healthy, injured, and aged skeletal muscles. Progress in the development of engineered cell delivery matrices designed for skeletal muscle regeneration will be reviewed. Finally, design considerations for successful MuSC delivery and engraftment, as well as other factors to further improve the functional outcome will be discussed. The current review will primarily focus on biomaterials designed for cell delivery. The findings and conclusions referenced in this review are for rodent models, and not from human data, unless noted otherwise. Tissue engineering strategies of skeletal muscle constructs ex vivo will not be extensively covered here, as such advancements have been recently reviewed elsewhere [25,26].

Satellite cells

MuSCs were first identified and characterized in 1961 in an electron microscope study, and have been implicated to play a role in muscle regeneration [27]. Indeed, recent cell ablation and lineage tracing studies have demonstrated that MuSCs, which express the paired box protein 7 (Pax7) transcription factor when quiescent under resting conditions, are essential to skeletal muscle regeneration [28–31]. In addition, advances in MuSC purification techniques using fluorescence-activated cell sorting enabled transplantation of Pax7⁺ MuSCs to the injured intramuscular space, where Pax7⁺ mouse and human MuSC transplantation into mouse recipients enhances regeneration [14–16,21]. Collectively, these transplantation studies demonstrate the importance of MuSC participation in the muscle regeneration process.

Recent studies identified several signaling pathways and mechanisms for maintenance of MuSC quiescence and self-renewal, including expression of microRNA-489 [32], phosphorylation of translation initiation factor eIFα2 [33], increased angiopoietin-1/Tie-2 signaling [34], and expression of EZH2 [35]. In the aged mice, basal autophagy has been identified as a critical regulator for the preservation of MuSC quiescence and prevention of senescence [36]. Within the quiescent MuSC pool, approximately 90% of the quiescent MuSCs express Myf5 and are biased to myogenic differentiation while the rest (~10%) do not [37]. Here, arginine methyltransferase Carm1 methylates arginines of Pax7, and subsequently induces transcription of Myf5 upon asymmetric division [38]. More recently, a study utilizing transgenic Pax7-nuclear GFP mice revealed that MuSCs expressing higher levels of Pax7 are less likely to commit and exhibit lower metabolic activity compared to MuSCs expressing lower levels of Pax7 [39], corroborating the presence of cell heterogeneity within the MuSC population.

With an onset of injury, MuSCs undergo activation, where the myoblast determination protein (MyoD) transcription factor is upregulated [40]. In presence of inflammatory cytokines such as IL-6, MuSCs commit to myogenic lineage progression in a STAT3-dependent manner [41]. One mechanism that regulates MyoD expression involves the histone H4 lysine 20 (H4K20) methyltransferase Suv4-20H, which binds the MyoD locus. When MuSCs are quiescent, H4K20 methyltransferase Suv4-20H1 is bound to the MyoD locus and maintains highly condensed heterochromatin structure [42]. Suv4-20H1 activity is reduced as a consequence of activation, exposing the MyoD locus from the condensed heterochromatin [42]. Furthermore, activated MuSCs undergo metabolic switch, which is characterized by decreased levels of NAD⁺ and SIRT1 deacetylase activity, subsequently leading to increased level of H4K16 acetylation [43]. This metabolic shift during activation impacts expression of MyoD expression [43]. MyoD⁺ myoblasts proliferate, migrate, and differentiate into myotubes upregulating myogenin (Myog) and downregulating Pax7 expression via p38/polycomb repressive complex 2 signaling [44–48]. In coordination with Notch signaling [49], Pax7⁻/Myog⁺ cells fuse to form multinucleated myotubes and subsequently new regenerating muscle fibers. A recent study identified myomaker, a muscle-specific membrane protein, as one of the key proteins that directly mediates the fusion process [50]. It is also important to note that the intrinsic function and number of MuSCs decline with aging, leading to inferior muscle regenerative capacity [7,51–57]. Comprehensive reviews on intrinsic changes in the MuSCs as a consequence of aging have been recently published [7,51,52].

Recent transplantation studies indicate that Pax7⁺ MuSCs represent a versatile cell source for treating muscle trauma, disorders, and age-associated muscle dysfunctions [14–16,21,53,58]. Transplantation of Pax7⁺ MuSCs to injured muscle results in rapid proliferation and engraftment resulting in muscle repair, but transplantation of cultured myoblasts exhibits limited capacity to induce muscle regeneration in vivo [15]. Furthermore, delivery of MuSCs to aged muscle reverses features of aging, including muscle mass, regenerative capacity, and contractile function [58]. Remarkably, delivery of MuSCs into dystrophin-deficient muscles of mdx mice also re-establishes dystrophin expression and enhances muscle function [16]. Embryonic stem and induced pluripotent stem cells that conditionally express Pax7 also result in effective restoration of dystrophin expression and muscle function over 11 months [59]. However, age-associated intrinsic function of MuSC is an important consideration for such translational applications, as the engraftment potential progressively declines with the cellular age [53,60]. Collectively, the evidence demonstrate that Pax7-expressing stem cells indeed are a promising candidate for treating muscle trauma, disorders, and age-associated muscle dysfunctions.

Satellite cell niche

MuSC function and the inherent regeneration capacity of skeletal muscle are facilitated by intricate interactions between MuSCs and their niche. The microenvironmental niche of MuSCs provides biomechanical and biochemical signals that regulate MuSC functions, including self-renewal, proliferation, differentiation, and migration [61]. MuSCs reside in between the sarcolemma of muscle fibers and the basal lamina matrix, where the basal lamina consists of an array of extracellular matrix proteins. In addition to providing biophysical cues to the cells, these matrix components also provide important sites for soluble growth factor sequestration and presentation [62,63]. Release of growth factors from the matrix, and other resident cells provide additional complexity for controlling MuSC function during homeostasis and repair. Identifying key niche components, and understanding mechanisms by which such factors control MuSC function, is an important engineering consideration for designing translatable and functional synthetic matrices.

Biomechanical signals are an important regulator for directing MuSC function. A seminal study by Gilbert et al. (2008) demonstrated that substrate elasticity is critical in regulating MuSC self-renewal function in vitro, where physiologically relevant stiffness (12 kPa) is essential for directing MuSC self-renewal in a 2D culture [20]. Whereas it remains to be determined whether this also extends to 3D culture systems, it is likely that the mechanical properties of the MuSC niche regulate MuSC function in vivo. For instance, collagen VI, a component of the basal lamina, plays an important role controlling MuSC self-renewal and muscle regeneration by providing structural and mechanical support to the niche [64]. Knockout of collagen VI results in a significantly decreased muscle elastic modulus, but restoration of collagen VI results in reestablishment in the tissue modulus, and more importantly, MuSC functions are rescued [64]. This suggests that any compositional changes arising from damage, aging, and pathology (e.g., muscular dystrophy, cachexia) could alter the physiological mechanical microenvironment, ultimately compromising the optimal MuSC function.

The basal lamina matrix is composed of extracellular matrix proteins, such as laminins, fibronectin, and collagens [65,66]. Notably, laminins are one of the major components of the basal lamina. MuSCs interact with laminins primarily via α7β1 integrin, which is specifically expressed in MuSCs [67,68]. α7β1 integrin binds to the E8 domain of laminin [69] and contributes to proliferation, adhesion, and migration properties [70–73]. Interestingly, mutations in α7 integrin result in congenital myopathies and muscular dystrophies, and furthermore, enhancement in α7 integrin expression delays the progression of muscular pathologies [74,75]. Injection of laminin into mouse models of congenital myopathies and eccentric exercise restores the regenerative capacity of the muscle [76–78], suggesting that interactions between α7 integrin and laminins are essential for the normal physiology of the muscle. Similarly, MuSCs lacking dystroglycan, a laminin receptor, exhibit decreased regenerative capacity [79], and blocking MuSC α6 integrin via RNA interference, thereby inhibiting interaction with the E8 domain of laminin, prevents myogenic differentiation [80], emphasizing the critical role that laminins play in directing MuSC function.

Fibronectin is another key component of the basal lamina that contributes to the regulation of MuSC function. In resting muscle, MuSCs reside in a close proximity to fibronectin-rich areas [81]. Indeed, MuSCs express syndecan-4 and Frizzled-7 co-receptor complexes that bind to fibronectin, where subsequent interactions between the complex and fibronectin promote symmetric division of MuSCs in the presence of Wnt7a [81]. Interestingly, fibronectin expression transiently increases with an onset of injury, and this transient expression induces Wnt7a signaling via syndecan-4 and Frizzled-7 co-receptor complex to expand and maintain the MuSC pool [81]. Furthermore, activated MuSCs gain β3 integrin expression, which interacts with the Arg-Gly-Asp (RGD) motif of fibronectin, and regulates myogenic differentiation and muscle regeneration [82–84]. Activated MuSCs also gain αV and α5 integrin expression [73], which complex with β3 and β1 integrin respectively to interact with the RGD motif of fibronectin. This data suggest that fibronectin is a key extracellular matrix protein involved in the orchestration of skeletal muscle regeneration.

Proteoglycans, such as perlecan, decorin, and biglycans, are also components of the basal lamina matrix [61]. While it remains to be determined whether proteoglycans directly regulate MuSC function, proteoglycans certainly modulate the MuSC niche in an indirect manner. For instance, perlecan-deficient mice exhibit muscle hypertrophy in a myostatin-dependent manner, suggesting that perlecan is crucial for maintaining muscle mass [85]. Furthermore, proteoglycans sequester important soluble growth factors to mediate MuSC function [62,63], such as insulin-like growth factor-1 (IGF-1), hepatocyte growth factor-1 (HGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF- β), and platelet-derived growth factor (PDGF-1). Specifically, such sequestration of growth factors can control the local concentration by releasing the growth factors “on-demand” upon mechanical perturbation.

Inferior regenerative capacity and progression of sarcopenia in aged muscle are due to changes in the MuSC niche [86] and declines in intrinsic MuSC function [7] that occur with aging. A recent study demonstrated that the local microenvironmental stiffness (measured via Atomic Force Microscopy (AFM)) increases with aging [87]. The increased tissue stiffness is attributed to accumulation of advanced glycation end-products and collagen [87]. Ultimately, progressive accumulation of fibrotic matrix proteins leads to chronic fibrosis within the muscle, and results in impaired regeneration [7,89] and progression of sarcopenia [86]. In addition to increased fibrotic tissue accumulation, aged muscles also exhibit impaired expression of Notch ligand Delta on the sarcolemma [90], where Notch signaling regulates critical functions of MuSCs, and therefore, muscle regeneration [91,92]. Finally, systemic alterations in Wnt3a, oxytocin hormone, and IL-6 have also been suggested to impact the muscle regeneration capacity with aging [93–95]. Restoration and augmentation of key niche elements that change as a function of aging through provisional matrices may provide an effective strategy to reinstate the skeletal muscle function upon onset of injuries and age-associated pathology in the aged skeletal muscle.

Roles of inflammation and fibroadipogenic progenitor cells (FAPs) in skeletal muscle regeneration

Inflammation is a critical process for adequate muscle regeneration following injury, which is characterized by massive infiltration of macrophages and neutrophils [96]. The initial phase of inflammation involves invasion of neutrophils and macrophages and M1 macrophage activation (Figure 1A and B) [96,97]. Neutrophils remove necrotic tissues and debris at the site of injury. Activated M1 macrophages secrete various pro-inflammatory signals, including IL-1β, IL-12, reactive oxygen species, nitric oxide, as well as insulin-like growth factor 1, which initiate MuSC activation and proliferation (Figure 1A) [96,98,99]. At this stage, fibroadipogenic progenitor cells (FAPs), resident myofibroblasts responsible for laying down transient extracellular matrix for myoblast proliferation and differentiation, are activated (Figure 1A) [18,89]. In rodent muscles, activated FAPs are cleared after ~96 hours post injury, where infiltrating macrophages induce FAP apoptosis via tumor necrosis factor α (TNF-α) [89]. Gradually, the pro-inflammatory M1 macrophages transition into constructive, wound-healing M2 macrophages that promote regeneration and resolution of inflammation through secretion of anti-inflammatory (e.g., IL-10, TGF- β), myogenic (e.g., IGF-1), and angiogenic (e.g., VEGF) factors (Figure 1B) [100–105].

Figure 1.

(a) Immediate response to muscle injury. Upon injury, quiescent muscle satellite cells (MuSCs) activate, self-renew, proliferate, and differentiate into myoblasts. Fibroadipogenic progenitors (FAPs) also activate, differentiate, and subsequently secrete transient extracellular matrix. Neutrophils infiltrate the site of injury and remove debris. (b) Regeneration in young niche. Pro-inflammatory M1 macrophages induce FAP apoptosis and recruit neutrophils. Anti-inflammatory M2 macrophages secrete anti-inflammatory, myogenic, and angiogenic factors. Myoblasts differentiate and fuse to form new muscle fibers. (c) Regeneration in aged/pathologic niche. MuSC niche composition is altered and results in stiffening. Normal inflammatory response is dysregulated, where inflammation persists over a longer period of time. FAPs over-secrete extracellular matrix, leading to progressive fibrosis. Myogenesis is impaired.

In the context of chronic muscle injury and aging, coordination of inflammatory cells, FAPs, and MuSCs is disrupted (Figure 1C). For instance, a recent study reported that regulatory T cell accumulation is significantly diminished in aged muscle, which subsequently impacts the activity of IL-33-expressing FAPs [106]. In muscles with chronic injury, apoptosis of FAPs is dysregulated due to elevated levels of TGF-β-producing macrophages, resulting in fibrotic degeneration of the muscle (Figure 1C) [89]. This further highlights that inflammation plays an important role in regulating the MuSC niche, which ultimately governs the regenerative outcome.

Delivery matrices for MuSC engraftment and function

Delivery of Pax7⁺ MuSCs is an effective strategy to stimulate and accelerate skeletal muscle regeneration, and therefore, to treat muscle injuries [14–16]. However, direct delivery of MuSCs via bolus injection exposes the transplanted cells to a harsh inflammatory environment and compromises their long term viability and function. Furthermore, transplanted MuSCs exhibit limited migration in vivo and thus rescue only a partial muscle volume, significantly limiting their therapeutic efficiency. A potential solution to overcome these hurdles is utilization of delivery matrices that provide protection and guide MuSC function (Figure 2a). In particular, hydrogels exhibit numerous advantageous properties as protein and cell delivery vehicles. For example, hydrogels can be injected in a minimally invasive manner, allow efficient nutrient exchange, and can be tuned to incorporate desirable structural, biophysical, and biochemical cues. Furthermore, such cell-delivery platforms also provide an opportunity to spatiotemporally present important MuSC niche elements that represent the asymmetry of matrix and adhesive components that modulate MuSC function, as MuSCs are exposed to biphasic interactions on the basal side of the matrix and on the apical side of muscle fiber sarcolemma. In this section, hydrogel-based biomaterials, both natural and synthetic, that have been developed to deliver skeletal muscle cells will be reviewed.

Figure 2.

(a) Biomaterial-mediated regeneration. Upon injury, quiescent muscle satellite cells are delivered within a biomaterial. Biomaterial encapsulation provides initial protection of the transplanted cells from inflammation. (b) Biomaterial promotes cell survival by providing integrin attachment sites. Donor cells within the biomaterial activate, self-renew, proliferate, migrate, and differentiate to induce host muscle regeneration. Encapsulated growth factor is released in a controlled manner, as the biomaterial is degraded by the cell-secreted proteases.

Collagen is a naturally derived material that has been used to examine the function of myoblasts. Collagen gels are bioactive, where cells can recognize inherently available cell-binding ligands. Indeed, encapsulating C2C12 or primary skeletal myoblasts in collagen gels promotes proliferation, differentiation, and fusion to form multi-nucleated myotubes in vitro [107–111]. Studies using collagen gels have set an important foundation for studying skeletal muscle cell function and early muscle development in a 3D context. However, there are several limitations associated with collagen gels to be used as a cell delivery vehicle. One limitation is that the major component of the MuSC niche is laminin [65,66], and that MuSC express laminin-specific integrins [67,68,80]. Indeed, culturing MuSCs in Matrigel, solubilized basement membrane protein rich in laminin, led to higher number of Pax7⁺ and MyoD⁺ cells than in collagen gels [112]. Furthermore, differentiated cells in laminin-rich Matrigel also resulted in more and larger MyoG⁺ myotubes than in collagen gels [112], highlighting the importance of the MuSC interaction with the underlying matrix. Here, optimizing the material interaction with MuSCs, rather than myoblasts, is important in the stem cell therapy context, because delivery of myoblasts results in suboptimal engraftment over time compared to MuSCs [15,113]. In particular, replenishing quiescent and self-renewing pool of MuSCs through biomaterials would be important for subsequent rounds of regeneration. Another drawback of using collagen gels is inability to modulate cell adhesive ligands and spatiotemporally present them to both the encapsulated and endogenous cells, which may be powerful tools to precisely control MuSCs.

Alginate is a natural biomaterial derived from brown algae. Alginate gels can be chemically modified to incorporate cell adhesive ligands and to exhibit varying range of stiffness and degradability [114]. Furthermore, due to their cytocompatibility and low toxicity, these materials have been extensively used in the biomedical applications [114]. For example, encapsulated myoblasts in RGD-conjugated alginate gels can proliferate, migrate and fuse into multi-nucleated myotubes in a ligand density-dependent manner in vitro [84,115,116]. Here, outward migration rate of the cells was largely dependent on the structural characteristics of the hydrogel [116]. Furthermore, co-delivery of myoblasts with HGF/FGF2 or VEGF/IGF in RGD-containing alginate gels to injured muscles accelerated regeneration with enhanced muscle mass and contractile function compared to delivering the cells alone [117–119]. It is also important to note that these studies utilized primary myoblasts that have been expanded on tissue culture dishes. Culturing muscle progenitor cells on stiff tissue culture plastics typically abolishes their stemness and results in inefficient engraftment [15,20], and therefore, it is not yet clear how these systems would perform if MuSCs were delivered to the injured muscles. Nonetheless, these investigations have demonstrated that delivery of growth factors in an injury-dependent context (e.g., VEGF/IGF for ischemic/myotoxin injury and HGF/FGF2 for laceration injury) and presentation of cell-adhesive ligands, such as RGD, are effective strategies for augmenting skeletal muscle regeneration.

Hyaluronic acid (HA), an anionic, non-sulfated glycosaminoglycan component of the extracellular matrix, is compatible with myoblasts, suggesting HA gels as a potential vehicle for MuSC delivery [120]. Indeed, delivery of MuSC via UV-crosslinked HA hydrogels to surgically ablated rodent muscles results in enhanced cell engraftment and functional muscle regeneration compared to delivery of myoblasts in HA hydrogel or hydrogel alone [113]. It is important to note that UV-mediated crosslinking is limited due to light scattering and attenuation in biological tissues from a translational perspective. Furthermore, despite HA’s cytocompatibility, most HA-based hydrogels are subject to degradation by hyaluronidases in vivo, where modulation of HA degradation may compromise the desired structural and mechanical properties of the HA hydrogels without proper chemical modification. While the effects of material degradation rate on skeletal muscle regeneration have not been evaluated, it is likely that premature and uncontrolled degradation may expose donor cells to neutrophils and macrophages post-injury, where the inflammation profiles may change with aging and pathologic degeneration. Furthermore, uncontrolled material degradation may induce premature donor cell anoikis - apoptosis induced from loss of cellular attachment to the material [121].

In the context of skeletal muscle engineering, fibrin gels created from fibrinogen and thrombin also have been explored [122–124]. Due to relatively rapid polymerization process and tunabilty of network structure, fibrin has been used in various biomedical applications, including skeletal muscle tissue engineering [125,126]. For instance, delivery of primary myoblasts to the rat skeletal muscle using fibrin gel results in donor cells fusing with the host muscle in a time-dependent and localized manner [122]. Furthermore, cultured myoblast/fibrin constructs can generate contractile force when electrically stimulated [123]. More recently, human muscle cell-seeded fibrin microthreads delivered to injured muscles of immuno-compromised mice induced skeletal muscle regeneration [124]. However, fibrin gels are not without their limitations. One major drawback is that fibrin gels formed at high concentrations exhibit extremely dense microstructure, where cellular infiltration and migratory behavior, as well as nutrient exchange, would be severely obstructed [126]. At low concentrations, fibrin gels become soft and rapidly lose their mechanical integrity. To overcome this limitation, a recent study incorporated gelatin beads into fibrin gels, where subsequent dissolution introduced micron-sized pores [127]. Introduction of macropores within the construct led to improved cell viability and myogenic differentiation of human umbilical cord mesenchymal stem cells [127], demonstrating the importance of material porosity on the cellular function and regenerative outcome. Similar to collagen gels, fibrin gels also contain numerous cell-binding sites for integrins [126], which is another limitation if a systematic approach to build provisional matrix for MuSCs is desired.

Poly(ethylene glycol) (PEG)-based synthetic hydrogels are highly cytocompatible, easy to manipulate chemically in order to alter structure, mechanical properties and the presentation of bioactive molecules, and exhibit non-fouling properties, and thus have been used extensively in biomedical applications. Synthetic materials, such as PEG, also are more advantageous than many naturally-derived materials due to diminished risk of pathogen transmission and lot-to-lot variability. Recently, a number of investigations have utilized PEG-based scaffolds (PEG-fibrinogen constructs) to deliver skeletal muscle-derived pericytes [128] and mesoangioblasts [129,130], and demonstrated that cell delivery in such materials promotes myogenic differentiation and skeletal muscle regeneration. Furthermore, controlled delivery of IGF-1 and SDF-1 using PEG-fibrin hydrogels also contributes to skeletal muscle regeneration [131,132]. In order to gain more precise control over building the cellular niche, PEG hydrogels that utilize maleimide crosslinking reaction chemistry also have been explored to culture C2C12 myoblasts in 3D [83]. Here, maleimide reactive groups enable conjugation of desired peptides (e.g., RGD and protease-degradable crosslinking peptides) to the PEG backbone in a highly specific manner. By systematically modulating the characteristics of this modular hydrogel system, myoblast viability and differentiation were found to be dependent on cell seeding density, polymer density, and bioadhesive ligands in 3D culture [83]. The resulting construct containing differentiated myoblasts also exhibited bulk contraction in vitro [83]. Further development and application of MuSC niche-containing synthetic biomaterials remains to be investigated.

Outlook

Recent advances in biomaterials demonstrate that engineered provisional matrices for MuSC may be a promising solution to improve long term regenerative outcome of stem cell therapy of muscle injuries and disorders. While the majority of previous investigations have focused on developing biomaterials for primary myoblasts, the C2C12 myoblast cell line, and other muscle-relevant cells (e.g., pericytes), biomaterials engineered to mimic the MuSC niche remain to be developed. Ideally, transplantation of MuSCs in such engineered biomaterial systems would protect the donor cells from the harsh inflammatory cells and promote cell survival, proliferation and migration by providing integrin attachment sites (Figure 2A). As inflammation resolves over time, the engineered matrix should provide biochemical (e.g., cell adhesive ligands, growth factors) and biophysical (e.g., matrix stiffness) cues necessary for the donor MuSCs to activate, self-renew and proliferate, differentiation, and eventually migrate out towards the sites of injury (Figure 2B). In particular, identification of niche-relevant cell-adhesive ligands (e.g., cell-binding peptides found in laminin, fibronectin, and collagen), integrins/receptors, as well as optimal ligand density that are necessary for MuSC survival and migration will be crucial for developing biomaterial-based approaches to overcome limitations associated with MuSC transplantation. Furthermore, co-delivery of growth factors, such as FGF-2, IGF-1, VEGF, and HGF-1, and other support cell types, such as FAPs, to promote survival, proliferation, differentiation, and migratory behaviors of the donor MuSCs needs to be investigated.

Biomaterial-based cell therapy approaches provide promising opportunities to spatiotemporally present ligands and growth factors to the transplanted cells. In the context of skeletal muscle regeneration, temporal coordination of MuSC expansion, commitment to myogenic progenitors, and differentiation is critical for biomaterial-mediated tissue repair. One possible approach would be to covalently functionalize a combination of caged- (i.e., contains protective chemical group that renders adhesive peptide inactive) and uncaged/active cell-adhesive peptides to the matrices [133]. For example, donor cells could be instructed to proliferate in the presence of uncaged/active α7β1-binding peptides, but directed to myogenic lineage and differentiate upon uncaging αvβ3-binding peptides [82], such as RGD. Presentation of spatially asymmetric provisional matrix, which mimic the physiologic MuSC niche consisting of basal lamina and myofibers-associated components, to the transplanted MuSCs may also be a productive strategy to improve the regenerative outcome. Furthermore, donor cells may also be co-delivered with multiple growth factors that release at varying rates for more effective therapeutic outcome. For instance, factors known to promote MuSC expansion at early time point upon transplantation, such as FGF2 or Prmt5 [134,135], and factors that promote vascularization, such as VEGF [136], can be incorporated into the system to be released at specific rates via a number of strategies, including direct non-covalent encapsulation in the bulk material, encapsulation in microspheres (sustained/delayed release), and covalently immobilizing (continuous/on-demand release) to the material [137]. For this strategy, material-specific control of growth factor release rate will be crucial for successful therapeutic outcomes.

Incorporating cell-demanded, protease-degradable cross-linkers that degrade the material in complement to hydrolytic degradation may also be an important consideration for accelerated MuSC engraftment and tissue regeneration (Figure 2B). Indeed, several types of matrix metalloproteinases (MMPs)-degradable cross-linkers have been previously implemented in synthetic matrices [138,139], where cell-secreted MMPs control the degradation of the material during proliferation, migration, and extracellular matrix synthesis. By using a combination of one or more cross-linkers that exhibit different degree of MMP-sensitivity, the rate of biomaterial degradation and matrix deposition may be more finely balanced, and ultimately enhance tissue regeneration.

Finally, development of engineered provisional matrices for MuSCs may also serve as a powerful tool for elucidating the interactions of MuSC and components of their niche in 3D. Such findings would not only be significant at the basic science level, but also would be critical for informing the development pathology-specific (e.g., traumatic injury, age-associated degeneration, muscular dystrophies) biomaterial-based therapies.

Highlights.

• Regenerative capacity of skeletal muscle declines with aging.

• Satellite cell delivery enhances regeneration in aging muscles.

• Direct delivery of satellite cells presents challenges.

• Advances in satellite cell and their niche biology are reviewed.

• Advances engineered cell delivery matrices for skeletal muscle are reviewed.