Nanomaterial for Skeletal Muscle Regeneration

Abstract

Skeletal muscle has an innate regenerative capacity to restore their structure and function following acute damages and injuries. However, in congenital muscular dystrophies, large volumetric muscle loss, cachexia, or aging, the declined regenerative capacity of skeletal muscle results in muscle wasting and functional impairment. Recent studies indicate that muscle mass and function are closely correlated with morbidity and mortality due to the large volume and location of skeletal muscle. However, the options for treating neuromuscular disorders are limited. Biomedical engineering strategies such as nanotechnologies have been implemented to address this issue.

In this review, we focus on recent studies leveraging nano-sized materials for regeneration of skeletal muscle. We look at skeletal muscle pathologies and describe various proof-of-concept and pre-clinical studies that have used nanomaterials, with a focus on how nano-sized materials can be used for skeletal muscle regeneration depending on material dimensionality.

Depending on the dimensionality of nano-sized materials, their application have been changed because of their different physical and biochemical properties.

Nanomaterials have been spotlighted as a great candidate for addressing the unmet needs of regenerative medicine. Nanomaterials could be applied to several types of tissues and diseases along with the unique characteristics of nanomaterials. However, when confined to muscle tissue, the targets of nanomaterial applications are limited and can be extended in future research.

Introduction

Skeletal muscle, occupying more than 40% of the human body weight, is the largest organ in the human body and plays an important role in movement, heat generation, and protection of internal organs [1]. Under normal physiological conditions, healthy adult skeletal muscle exhibits a robust ability to regenerate their structure and function following acute damages, physical injuries, and perturbations from wear and tear [2, 3]. This innate regenerative capacity is bestowed by highly orchestrated cellular and molecular processes between resident muscle stem cells (MuSC), also known as satellite cells, and their niche [1]. Even after catastrophic injuries, skeletal muscles initiate the adult myogenic process, which reconstructs vascular and neuronal networks and allows for the restoration of fully functional motor units. However, in congenital muscular dystrophies, severe compound injuries, large volumetric muscle loss, cachexia, or aging, the regenerative capacity of MuSCs progressively declines. Tissue fibrosis with chronic inflammation further exacerbates muscle wasting, followed by functional impairment [4]. Furthermore, due to the large volume and location of skeletal muscle, a growing body of research indicates that muscle mass and function are closely correlated with morbidity and mortality [5].

Since the discovery of satellite cells and recent advances in cellular and molecular analysis techniques, our understanding of muscle stem cell biology and cell-based therapy in regenerative medicine has significantly expanded [6, 7]. Despite the wealth of knowledge and the growing number of pre- and clinical studies, stem cell- based therapies are not currently being widely used in clinical setting due to several major limitations. These limitations include donor cell to patient incompatibility and immune rejections, limited functional benefits, and cancer formation. In order to overcome some of these hurdles, biomedical engineering strategies have been implemented. In the past decade, several innovative tissue engineering and nano-technologies have provided new approaches to improve targeted-drug delivery, biomimetic engineering of stem cell niche, or bioprinting of tissue microenvironment, and enhancing survival and differentiation of donor cells to boost long-term tissue function.

Nano-sized materials or nanomaterials can be defined as substances that have at least one dimension that is less than 100 nm in size [8]. Based on their size and shape, nanomaterials show unique and different physical and chemical properties compared to bulk materials that are composed of the same elements. Depending on the shape and size of nano-sized dimensions, nanomaterials can be classified into four dimensionality types (zero to three dimensional nanomaterials). The hierarchical and anisotropic structure of skeletal muscle, as well as the microenvironment of skeletal muscle cells, implicate the capability of all four types of nano-sized material application on skeletal muscle disease. When compared with traditional clinical approaches for skeletal muscle regeneration (e.g., interventions including surgical treatment and drug treatment), because of their unique properties, nano-sized materials are anticipated to become promising tools to overcome biological limitations and address unmet needs of skeletal muscle regeneration.

In this mini review, we focus on recent studies leveraging nano-sized materials for regeneration of skeletal muscle. We examine skeletal muscle pathology and summarize some of the proof-of-concept and pre-clinical studies using nanomaterials with specific emphasis on how nano-sized materials can be utilized for skeletal muscle regeneration based on the material dimensionality. Furthermore, future directions for enhancing therapeutic efficacy of nano-sized material-based treatments and some of the outstanding questions will be discussed.

Nano-sized materials for regenerative medicine

Nano-sized materials can be classified according to the shapes and sizes of their nano-sized dimensions [8]. Nanoparticles and quantum dots are zero-dimensional nanomaterials, nanorods and nanotubes are one-dimensional nanomaterials, nanofilms and nanolayers are two-dimensional nanomaterials, and nanocomposites are three-dimensional nanomaterials [8]. Nano-sized materials which have all four dimensionality types are utilized in the biomedical engineering field. Inorganic nanoparticles, organic polymer-based nanoparticles and extracellular vesicles are examples of zero-dimensional nanomaterials. These materials are used for drug delivery [9–12], imaging [13], and therapy itself [14–16]. Carbon nanotubes are considered one-dimensional nanomaterials, and graphene oxide is a two-dimensional nanomaterial. Because of electrochemical properties of carbon-based nanomaterials, carbon nanotubes and graphene oxide (GO) were utilized as therapeutic molecule carriers [17, 18] and enhancers for cell attachment [19]. Nanoparticles or graphene oxide decorated polymer scaffolds are examples of nanocomposites, classified under three-dimensional nanomaterials. These scaffolds were used for treatment of tissue regeneration [20]. Depending on the disease or target tissue, nanomaterials can offer appropriate mechanical, electrochemical, and biological properties to escalate the efficacy of treatment.

Nanostructures in the skeletal muscle

Inside the skeletal muscle, muscle fibers are compartmentalized with connective tissues. Fascicles, the organized bundles of myofibers, are surrounded by the connective tissue perimysium. Each myofiber inside the fascicles is enclosed with a thin layer of connective tissue called endomysium. Myofibers have diameters ranging between 20 and 100 µm and a collection of multiple myofibrils (Fig. 1A). The myofibrils are approximately 1.2 µm in diameter and are composed of a serial connection of sarcomeres [21]. Sarcomeres, the smallest functional units of a skeletal muscle fiber, have an arrangement of contractile and structural proteins essential for generating the motion of muscle fibers. Therefore, the anatomical structure of skeletal muscle can be considered as a hierarchical complex with organized sub-micron sized self-contracting functional units. These hierarchical and repeated structural characteristics of skeletal muscle tissue imply an application of nano-sized materials for new therapeutic regeneration approaches.

Fig. 1.

Hierarchical structure of muscle and muscle stem cell (MuSC) niche. A Structure of muscle anatomy. B Illustration of MuSC niche. MuSCs are tightly packed within the muscle fiber and basal lamina membrane. The interaction of muscle fiber (cell–cell interaction) and with extracellular matrix (ECM) of basal laminar (cell-ECM interaction) contribute to the regulation of MuSC cell cycle. The surrounding blood vessel, neuro muscular junction, fibroadipogenic progenitors and macrophage also attribute to MuSC niche

In addition to these structural features, skeletal muscle tissues have highly organized networks of connective tissue, nerves, blood vessels and myofibers. Each myofiber is enveloped with the basal lamina; the dense structure consists of basement membrane proteins such as laminin, fibronectin, and collagen type IV. Additionally, sulfated glycosaminoglycans, proteoglycans and collagen type I contribute to maintaining the integrity of skeletal muscle architecture [22, 23]. These extracellular matrix (ECM) proteins form nano-sized topography and fibrous structures that were observed in decellularized muscle ECM [24, 25]. Muscle stem cells, also known as satellite cells, are internal muscle tissue stem cells that exist in the space between the basal lamina and the sarcolemma of myofibers (Fig. 1B). During the skeletal muscle regeneration, MuSCs proliferate and differentiate directly into myofibers to regenerate functional tissue. Components of the muscle stem cell niche, such as ECM proteins, muscle fibers, blood vessels, neuromuscular junctions, mesenchymal stromal cells like fibro-adipogenic progenitors, and tissue resident immune cells play essential roles for the activation of MuSCs. The activated MuSCs enter into the cell cycle and proliferate to create a sufficient amount of progeny cells that differentiate into multinucleated myotubes to regenerate damaged tissue. Throughout the entire muscle regeneration process, communication between MuSCs and other components of the stem cell niche can be a significant target for biomedical engineering to improve the regenerative functions of skeletal muscle tissue. In the aspect of signaling communication during skeletal muscle regeneration, nano-sized materials can provide novel methods for efficiently delivering signaling molecules and drugs and enhance therapeutic applications [26].

Muscle pathology and traumatic muscle injuries

Traumatic injury, surgical loss of muscle, and congenital genetic alterations are the most common human muscular disorders. Volumetric muscle loss (VML) is defined as the loss of large amounts of muscle tissue caused by traumatic damage from various circumstances such as traffic accidents, military combats, and medical interventions. Generally, VML occurs with the critical loss of ECM, blood vessels and neuronal connection between skeletal muscle tissues. Therefore, innate regeneration processes of skeletal muscle tissues are impaired, and excessive fibrotic tissues replace the damaged muscle. As a result of VML, patients suffer from a deficiency of muscle function, which hinders them from daily activities and interactions. Additionally, limited treatment options available for VML make it a burden to society. Clinical and preclinical research have been investigated on cell-based approaches to treat VML; however, insufficient cell sources, low cell viability and regulatory issues diminish the therapeutic effect of treatments [27–30].

Duchenne’s muscular dystrophy (DMD) is the most common congenital muscular dystrophy caused by genetic alterations (deletion) of dystrophin protein. Dystrophin plays a role in skeletal muscle by connecting intracellular cytoskeleton to outer-cellular ECM through the binding of F-actin and dystrophin-associated protein complex [31]. The loss of dystrophin protein provokes downregulated expression of the dystrophin-associated proteins which induces continuous fiber damage and membrane leakage. The life expectancy of DMD patients is around 20 years due to respiratory failure symptoms that are developed in their late teenage years [32]. Although corticosteroids are used for standard care of DMD in the United States, it can be neither be a permanent nor the most effective treatment for DMD due to the drug’s side effects [4]. Recently, stem cell transplantation, therapeutic gene delivery, and small molecule delivery have been introduced for treatment of DMD. Even so, finding the most successful treatment for DMD is challenging because of the difficulty in delivering enough therapeutic agent to sufficient mass [32].

Nano-sized material for skeletal muscle regeneration

In biomedical engineering, nano-sized materials have been utilized for regeneration of nearly every tissue [33]. In terms of skeletal muscle regeneration, nano-sized materials can offer the promotion of cell migration and alignment, cell differentiation, biocompatibility, conductivity, and drug delivery properties. When combined with other bioengineering technologies, these distinct properties of nano-sized materials make it possible to fulfill unmet needs for the treatment of skeletal muscle disorders. The following sections will cover the concurrent application of nano-sized materials for skeletal muscle regeneration based on their dimensional types.

Nanoparticles

Nanoparticles such as inorganic, organic nanoparticles, and extracellular nano vesicles are considered zero-dimensional nanomaterials. On account of the small size, large surface area, capability of surface modification and drug loading capacity, nanoparticles are mainly used for therapeutic drug delivery [34–36]. In recent years, extracellular vesicles and lipid nanoparticles have been in the spotlight for their biocompatibility, simplicity of their formation, great bioavailability, and the ability to carry broad range of cargos [37].

Gold nanoparticle (AuNP) is the most common inorganic nanoparticle used in biomedical applications because of their decent biocompatibility and binding affinity with proteins [38]. Treatment of monodispersed AuNP and gold-silver alloy nanoparticles have been reported to enhance myoblast cell line (C2C12) attachment, proliferation, and differentiation through p38 mitogen-activated protein kinase (MAPK) signaling pathway [39]. Additionally, AuNP and gold-silver alloy nanoparticles showed promotion of in vivo skeletal muscle regeneration in tibialis anterior (TA) muscle defect model in rats. In other studies, AuNP has been utilized as a therapeutic protein delivery method for treatment of DMD. Immune-regulatory protein, interleukin-4 (IL-4) or interleukin-10 (IL-10) conjugated AuNP have shown improved muscle function in murine DMD model [40]. In this study, microdamage was applied to the TA of DMD mice to mimic chronic damage of DMD patient muscle. IL-4 or IL-10 loaded AuNPs were delivered to the TA via intramuscular injection. Fourteen days after AuNP injection, the recipient group with IL-4 conjugated AuNP showed improved muscle fiber area and muscle strength through upregulation of helper T cells and regulatory T cells. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system with therapeutic gene loaded AuNP have been developed for treatment of DMD [41]. After intramuscular delivery of the CRISPR-Cas9 system loaded AuNP, homology-directed repair occurred in the TA muscle, and the dystrophin protein was expressed in the TA without any elevation of systematic inflammatory cytokines. Along with AuNP, there are other various options of nanoparticle forming elements such as selenium [42], titanium decorated iron [43], and silica [44]. Despite the positive outcomes utilized for skeletal muscle regeneration, their limited flexibility for modification and biocompatibility compared to AuNP diminishes the application of nanoparticles with diverse elements.

Polymeric nanoparticles can be synthetized using diverse techniques such as microfluidics, emulsification, and nanoprecipitation [37]. The flexibility of preparation method allows polymeric nanoparticles to carry various type of cargos with different chemical properties and molecular weights. Therefore, both hydrophilic and hydrophobic drugs can be loaded in the nanoparticle core, polymer matrix, and nanoparticle surfaces [45]. Poly(lactic-co-glycolic acid) (PLGA) is one of the most widely used polymer for the synthesis of nanoparticles. Skeletal muscle targeted delivery of phosphatase and tension homolog (PTEN) inhibitor loaded PLGA nanoparticles for DMD treatment has been reported [26]. For the fabrication of nanoparticles, poly(ethylene glycol) functionalized (PEGylated) PLGA polymers were dissolved in an organic solvent and a therapeutic drug, VO-OHpic (PTEN inhibitor), was dissolved in aqueous phase. An emulsion was then formed with a probe sonicator to create nanoparticles. For the conjugation of skeletal muscle targeting moiety (muscle homing peptide M12, RRQPPRSISSHP, which can preferentially bind to surface protein of muscle cells) was incubated with PLGA nanoparticles. As a result, PLGA nanoparticles showed selective uptake by muscle cells both in vitro and in vivo. Even though the authors showed controlled release of PTEN inhibitor from the nanoparticles, they did not present any therapeutic effects of the drug in vitro and in vivo. PLGA nanoparticles have also been used for the delivery of therapeutic small molecules [46]. In this study, forskolin and RepSox have been reported as key factors for direct reprogramming of certain population of primary dermal cells. These small molecules have been loaded in the PLGA nanoparticle along with the emulsification method. The drug loaded nanoparticles represented enhanced myogenic cell production from dermal cells in vitro and elevated muscle regeneration and function in the in vivo model of TA muscle injury.

Exosomes and lipid nanoparticles are representative examples of lipid-based nanoparticles. Lipid-based nanoparticles typically have the structure of internal aqueous subdivision wrapped with lipid bilayer. Characteristics of these nanoparticles include high bioavailability, biocompatibility, simple formulation process, payload flexibility and a wide range of functional modifications which provide worthwhile benefits to therapeutic drug delivery systems [47–49]. For skeletal muscle regeneration, exosomes collected from different cell sources have shown therapeutic effects. These exosomes resemble the features of the origin cells and contain large species of bioactive molecules such as micro-RNA (miRNA), DNA, and proteins. Mesenchymal stem cell (MSC)-derived exosomes have been highlighted and utilized to improve regeneration in various tissues [50]. Likewise, MSC-derived exosomes enhanced muscular differentiation in vitro and in vivo [51, 52]. For myogenic factors in MSC-derived exosomes, miRNAs (miR-1, miR-133, miR-206, and miR-494) are designated as key factors [52]. Exosomes isolated from human skeletal muscle cell [52], cardiosphere-derived cell [53], M2 macrophage [54], and placenta-derived MSCs [55] have also shown enhanced therapeutic efficacy for treatment of skeletal muscle disorder. Ning Ran et al. have reported myostatin pro-peptide expression in the exosome surface marker CD63 and intravascular delivery of these exosomes to DMD mice increased muscle mass and muscle function [56]. In this study, authors constructed CD63-myostatin propeptide expressing lentivirus and transfected it to NIH3T3 fibroblast to promote myostatin propeptide expressed on the surface of exosomes. This strategy has increased serum stability of propeptide and therapeutic efficacy of myostatin by taking advantage of exosomes as a drug delivering carrier.

Nanofiber

Polymeric nanofibers, composed of a wire or string structure with a nano sized diameter, are one-dimensional nanomaterial. Fiber structures are abundant in nature as seen in the human body and in the hierarchical architecture of the skeletal muscle. A major ECM protein in the skeletal muscle is collagen and it exists in a nanofibril form (10–300 nm diameters) [57]. To mimic this natural nanofiber structure, polymeric nanofibers have been fabricated via electrospinning (refer to [58] for detailed review about electrospinning). PLGA [59, 60], chitosan-conjugated polycaprolactone (PCL) [61], silk [61], cellulose nano-whiskers [62], and alginate/PCL [63] nanofibers with various nanoscale diameters are utilized for enhancing proliferation and differentiation of in vitro C2C12 myoblasts experiments. As an in vitro application of nanofibers, gelatin nanofibrous sheets have been fabricated with sacrificial coaxial electrospinning of PCL/gelatin solution [64]. After C2C12 seeding on the nanofiber sheet, a stepwise stretching step was applied to induce enhanced myotube formation on the nanofiber scaffold. The nanofiber/differentiated C2C12 construct implanted on the VML model with defects in quadriceps showed elevated muscle regeneration and weight bearing function of the limb.

Graphene oxide

GO is one of the graphene derivatives that have carboxyl (–OOH), epoxy (–O), and hydroxyl (–OH) functional groups on the basic hexagonal carbon lattice structure of graphene [65]. The morphology of GO is similar to very thin paper, considered as two-dimensional nanomaterial. The thickness of a monolayer of GO is around 0.7 nm, and it can be stacked via their interlayer interactions of the pi-bonding of graphene unit cells [66]. Not only does it display great physicochemical properties but GO also has significant biological properties such as biocompatibility, easy functional modification, strong affinity with biomolecules (nucleic acids and proteins), and stability in the biological solutions [67]. An interesting chemical property of GO is its ability to act as an electron acceptor due to the chemical configuration. When GO is introduced to a biological system (in the cell), GO is able to change the oxidative state and signaling pathways related to oxidative stress [68, 69]. Enhanced differentiation of skeletal muscle cultured on GO or reduced GO (rGO, containing less oxygen content than GO) has been investigated in several studies [70–72]. As an in vitro application of GO, a stretchable and transparent muscle cell-graphene hybrid was fabricated by Kim et al. [73]. By utilizing electroconductivity and bio functionality of GO, researchers created a cell-sheet-graphene hybrid that enables recording, stimulation, and therapy in the skeletal muscle tissue. C2C12 seeded on the device showed improved elongation and alignment according to the electrical pulse stimulation. After implantation of the device in the hindlimb, optogenetic stimulation enabled muscle movements and implanted muscle cells proliferated on the muscle tissue.

Nanomaterial composite

Nanomaterial composites denote combined materials which contains more than one nanomaterial [74, 75]. Even though all three dimensions are no longer nano-sized, nanomaterial composite can be considered as three-dimensional nanomaterials. By combining nanomaterials and other kind of materials, various physical, electrochemical, and biological properties can be combined and exploited to improve the therapeutic function of the materials.

The most common example of a nanomaterial composite is a nanoparticle or graphene oxide containing electrospun nanofibers [76–79]. GO-functionalized PLGA-collagen fibers showed enhanced mechanical properties and increased differentiation of C2C12 cells compared with bare polymer nanofibers [76]. PCL/silk fibroin nanofiber and poly(ethylene glycol) hydrogel composites were also reported to demonstrate increased alignment and differentiation of C2C12 cells in multilayers [80]. However, electrospun nanofibers were not used for in vivo studies on skeletal muscle regeneration. Rather, hydrogels incorporated with nanoparticles were used for in vitro and in vivo experiments. Ge et al. reported that gold nanoparticle containing injectable Pluronic F-127 hydrogel presented increased structural and functional regeneration in the VML mouse model [81]. Another application of nanomaterial composites is as an additive for 3D printing [82]. In this study, gold nanowires were simultaneously printed with C2C12 cells in collagen-based bio ink. Through the application of an electric field to the printed scaffold, gold nanowires were aligned in parallel and consequently increased C2C12 differentiation. When implanted to a volumetric muscle loss model, the cell laden hydrogel with aligned nanowires presented an increase in muscle regeneration and decreased in fibrotic area.

Conclusions and future directions

Nanomaterials have been spotlighted as a great candidate for addressing the unmet needs of regenerative medicine. Nanomaterials could be applied to several types of tissues and diseases along with the unique characteristics of nanomaterials [37]. However, when confined to muscle tissue, the targets of nanomaterial applications are limited and can be extended in future research. (Fig. 2).

Fig. 2.

Present and future targets for application of nanomaterial to skeletal muscle regeneration

Most of the studies used C2C12 myoblasts, which are not the same as primary cells, and the immortalized C2C12 cells is different from native microenvironment of skeletal muscle. To address this issue, researchers may use primarily isolated muscle stem cell or myoblast, or pluripotent stem cell derived myoblast as a cell source for in vitro and in vivo study. Skeletal muscle tissue regeneration is a complicated process that includes immune response, angiogenesis, and innervation. Although regulating the immune system during regeneration has been dealt by some studies, targeting muscle tissue resident macrophages and regulation of their phenotypes can be new therapeutic approaches. Angiogenesis and innervation are essential for regeneration of functional muscle tissue. Therefore, integrated strategies targeting reconstruction of blood vessel and motor neuron architecture in the skeletal muscle tissue harnessing features of nanomaterials can be suggested. As a therapeutic target cell type for nanomaterials, fibro/adipogenic progenitors (FAPs) have enormous opportunities. FAPs are muscle tissue resident mesenchymal stem cells, and it supports homeostasis and repair of skeletal muscle tissue. Even though FAPs are not muscle cells, targeting FAPs with nanomaterials can improve therapeutic efficacy of muscle regeneration. MuSC niche cannot be underestimated for therapeutic target. Nanomaterials can boost muscle regeneration by controlling MuSC niche in a delicate manner to regulate MuSC proliferation and differentiation behavior. Overall, nanomaterials contain promising possibilities for skeletal muscle regeneration due to their unique characteristics and various application methods.

Declarations

Conflict of interest

The authors have no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.