Skeletal Muscle Tissue Engineering Research Articles

Electrospinning Aligned SF/Magnetic Nanoparticles-Blend Nanofiber Scaffolds for Inducing Skeletal Myoblast Alignment and Differentiation.

In the realm of skeletal muscle tissue engineering, anisotropic materials that emulate natural tissues show substantial promise. Electrospun scaffolds, mimicking the fibrillar structure of the extracellular matrix, are commonly employed but often fall short in achieving optimal alignment and mechanical strength. Silk fibroin has emerged as a versatile material in tissue engineering, valued for its biocompatibility, mechanical robustness, and biodegradability. However, conventional electrospinning methods of SF result in randomly oriented fibers, limiting their efficacy. In this work, we developed a straightforward method to fabricate directional tissue scaffolds using silk fibroin. By integrating a magnetic field collecting device and incorporating Fe3O4 nanoparticles into the spinning solution, we successfully produced well-aligned silk nanofiber scaffolds. These aligned fibers not only improved scaffold orientation and mechanical properties but also exhibited magnetic responsiveness. The aligned SF scaffolds effectively guided the adhesion, proliferation, and differentiation of mesenchymal stem cells along the fiber direction. Cultured on these scaffolds, myoblast C2C12 cells demonstrated oriented growth, highlighting the potential of aligned SF fibers in advancing skeletal muscle engineering for biomedical applications.

Extrusion-Based 3D Bioprinting of Bioactive and Piezoelectric Scaffolds as Potential Therapy for Treating Critical Soft Tissue Wounds.

Objective: This study focuses on developing bioactive piezoelectric scaffolds that could deliver bioelectrical cues to potentially treat injuries to soft tissues such as skeletal muscles and promote active regeneration. Approach: To address the underexplored aspect of bioelectrical cues in skeletal muscle tissue engineering (SMTE), we developed piezoelectric bioink based on natural bioactive materials such as sodium alginate, gelatin, and chitosan. Extrusion-based 3D bioprinting was utilized to develop scaffolds that mimic muscle stiffness and generate electrical stimulation (E-stim) when subjected to forces. The biocompatibility of these scaffolds was tested with the C2C12 muscle cell line. Results: The bioink demonstrated suitable rheological properties for 3D bioprinting, resulting in high-resolution composite sodium alginate-gelatin-chitosan scaffolds with good structural fidelity. The scaffolds exhibited a 42-60 kPa stiffness, similar to muscle. When a controlled force of 5N was applied to the scaffolds at a constant frequency of 4 Hz, they generated electrical fields and impulses (charge), indicating their suitability as a stand-alone scaffold to generate E-stim and instill bioelectrical cues in the wound region. The cell viability and proliferation test results confirm the scaffold's biocompatibility with C2C12s and the benefit of piezoelectricity in promoting muscle cell growth kinetics. Our study indicates that our piezoelectric bioink and scaffolds offer promise as autonomous E-stim-generating regenerative therapy for SMTE. Innovation: A novel approach for treating skeletal muscle wounds was introduced by developing a bioactive electroactive scaffold capable of autonomously generating E-stim without stimulators and electrodes. This scaffold offers a unique approach to enhancing skeletal muscle regeneration through bioelectric cues, addressing a major gap in the SMTE, that is, fibrotic tissue formation due to delayed muscle regeneration. Conclusion: A piezoelectric scaffold was developed, providing a promising solution for promoting skeletal muscle regeneration. This development can potentially address skeletal muscle injuries and offers a unique approach to facilitating skeletal muscle wound healing.

Engineered Regenerative Isolated Peripheral Nerve Interface for Targeted Reinnervation.

A regenerative peripheral nerve interface (RPNI) offers a therapeutic solution for nerve injury through reconstruction of the target muscle. However, implanting a transected peripheral nerve into an autologous skeletal muscle graft in RPNI causes donor-site morbidity, highlighting the need for tissue-engineered skeletal muscle constructs. Here, an engineered regenerative isolated peripheral nerve interface (eRIPEN) is developed using 3D skeletal cell printing combined with direct electrospinning to create a nanofiber membrane envelop for host nerve implantation. In this in vivo study, after over 8 months of RPNI surgery, the eRIPEN exhibits a minimum Feret diameter of 15-20µm with a cross-sectional area of 100-500 µm2, representing the largest distribution of myofibers. Furthermore, neuromuscular junction formation and muscle contraction with a force of ≈28 N are observed. Notably, the decreased hypersensitivity to mechanical/thermal stimuli and an improved tibial functional index from -77 to -56 are found in the eRIPEN group. The present novel concept of eRIPEN paves the way for the utilization and application of tissue-engineered constructs in RPNI, ultimately realizing neuroprosthesis control through synaptic connections.

Biodegradable conductive IPN in situ cryogels with anisotropic microchannels and sequential delivery of dual-growth factors for skeletal muscle regeneration

Biodegradable and anisotropic cryogels that simulate conductivity and ECM orientation structure of skeletal muscle, and release multiple growth factors, are expected for in situ skeletal muscle tissue engineering. Herein, biodegradable, conductive and anisotropic interpenetrating network (IPN) in situ cryogels are fabricated through Schiff base/acylhydrazone crosslinking via combining unidirectional freezing and cyclic freeze-thaw processes. The cryogels have good anisotropic mechanical properties and oriented microchannel structure, and induce the oriented alignment of myoblasts. The introduction of aniline tetramer enhances the mechanical properties and conductivity of the cryogels. The conductive cryogels significantly improve the proliferation and myogenic differentiation of C2C12 cells during 3D culture. The sequential delivery of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) can induce migration of human umbilical vein endothelial cells (HUVECs), proliferation of human skin myofibroblasts (HMFB), and myogenic differentiation of C2C12 cells in vitro. In particular, conductive and anisotropic cryogels with dual-growth factors can significantly improve the repair efficiency of volumetric muscle loss (VML) in vivo. This study provides a new strategy to fabricate anisotropic and conductive IPN in situ cryogel biomimetic scaffolds that can encapsulate dual-growth factors and deliver them in time sequence, which significantly promote the efficient repair of VML via an in situ tissue engineering approach.

Impact of Passaging Primary Skeletal Muscle Cell Isolates on the Engineering of Skeletal Muscle.

Volumetric muscle loss (VML) is a clinical state that results in impaired skeletal muscle function. Engineered skeletal muscle can serve as a treatment for VML. Currently, large biopsies are required to achieve the cells necessary for the fabrication of engineered muscle, leading to donor-site morbidity. Amplification of cell numbers using cell passaging may increase the usefulness of a single muscle biopsy for engineering muscle tissue. In this study, we evaluated the impact of passaging cells obtained from donor muscle tissue by analyzing characteristics of in vitro cellular growth and tissue-engineered skeletal muscle unit (SMU) structure and function. Human skeletal muscle cell isolates from three separate donors (P0-Control) were compared with cells passaged once (P1), twice (P2), or three times (P3) by monitoring SMU force production and determining muscle content and structure using immunohistochemistry. Data indicated that passaging decreased the number of satellite cells and increased the population doubling time. P1 SMUs had slightly greater contractile force and P2 SMUs showed statistically significant greater force production compared with P0 SMUs with no change in SMU muscle content. In conclusion, human skeletal muscle cells can be passaged twice without negatively impacting SMU muscle content or contractile function, providing the opportunity to potentially create larger SMUs from smaller biopsies, thereby producing clinically relevant sized grafts to aid in VML repair.

Efficient expansion culture of bovine myogenic cells with differentiation capacity using muscle extract-supplemented medium

The production of tissue-engineered skeletal muscles for cell-based therapy and cultivated meat production requires a large number of myogenic cells capable of differentiating into myotubes. A suspension culture with a stirred bioreactor is a system that is feasibly scalable, but scaling up the production of myogenic cells while maintaining differentiation capacity requires an efficient culture technique. This remains challenging in suspension culture. On the other hand, conventional meat processing generates substantial by-products, including meat trimmings containing effective substances for myogenic cell cultures. Efficiently utilizing these by-products can reduce environmental impact and disposal costs. Here, we describe an expansion culture method for bovine myogenic cells (BMCs) using Dulbecco's Modified Eagle's medium (DMEM) supplemented with muscle extracts. During the 6-day planar culture, the proliferation rate using the culture medium supplemented with muscle extracts (CMME) and 10% fetal bovine serum (FBS) was 18 ± 3.3, three times higher than that achieved using DMEM with 10% FBS. Furthermore, a combination of CMME supplemented with 10% FBS and an iMatrix-511-coated dish could maintain differentiation capacity through passaging culture. Finally, we demonstrated a suspension culture with microcarriers using CMME with 10% FBS. Through 3 passages, the cumulative proliferation rate was 191.7 ± 35.1, four times higher than that of produced DMEM with 10% FBS. Using CMME, the cells maintained differentiation capacity in comparison to that using DMEM. This approach will contribute to both the reduction of wasted by-products from conventional meat production and the development of a suspension culture system for maintaining high-quality myogenic cells.

Biomaterials-Based Technologies in Skeletal Muscle Tissue Engineering.

For many clinically prevalent severe injuries, the inherent regenerative capacity of skeletal muscle remains inadequate. Skeletal muscle tissue engineering (SMTE) seeks to meet this clinical demand. With continuous progress in biomedicine and related technologies including micro/nanotechnology and 3D printing, numerous studies have uncovered various intrinsic mechanisms regulating skeletal muscle regeneration and developed tailored biomaterial systems based on these understandings. Here, the skeletal muscle structure and regeneration process are discussed and the diverse biomaterial systems derived from various technologies are explored in detail. Biomaterials serve not merely as local niches for cell growth, but also as scaffolds endowed with structural or physicochemical properties that provide tissue regenerative cues such as topographical, electrical, and mechanical signals. They can also act as delivery systems for stem cells and bioactive molecules that have been shown as key participants in endogenous repair cascades. To achieve bench-to-bedside translation, the typical effect enabled by biomaterial systems and the potential underlying molecular mechanisms are also summarized. Insights into the roles of biomaterials in SMTE from cellular and molecular perspectives are provided. Finally, perspectives on the advancement of SMTE are provided, for which gene therapy, exosomes, and hybrid biomaterials may hold promise to make important contributions.

Thickening tissue by thinning electrospun scaffolds for skeletal muscle tissue engineering

AbstractElectrospun scaffolds with aligned fiber orientation are widely used in tissue engineering, such as muscle, heart, nerve, tendon, and cartilage, due to their ability to guide cell morphology and induce cellular functions. However, the dense fibrous structure of the scaffolds poses a critical obstacle to engineering highly cellular and thick 3D tissues, as it prevents cell infiltration. While many techniques have been developed to increase the pore size of electrospun scaffolds and improve cell infiltration/migration, it often leads to a decrease in direct cell‐cell contact, compromising cell differentiation and tissue maturation. This study presents an alternative approach by reducing the thickness of scaffolds to the cellular scale and stacking or rolling the cell‐scaffold complex into 3D constructs. We devise a series of novel tools to fabricate, characterize, and manipulate ultra‐thin electrospun scaffolds, which demonstrate high reproducibility, resolution, and cellularity. Our study provides a solution to the cell infiltration issue in muscle tissue engineering and is highly versatile, and can be applied to various fields that require structures with high‐resolution gradients in a layered pattern or complex spatial distribution in a rolled pattern.

Decellularised plant scaffolds facilitate porcine skeletal muscle tissue engineering for cultivated meat biomanufacturing

Cultivated meat (CM) offers a sustainable and ethical alternative to conventional animal agriculture, involving cell maturation in a controlled environment. To emulate the structural complexity of traditional meat, the development of animal-free and edible scaffolds is crucial, providing vital physical and biological support during tissue development. The aligned vascular bundles of the decellularised asparagus scaffold were selected to facilitate the attachment and alignment of murine myoblasts (C2C12) and porcine adipose-derived mesenchymal stem cells (pADMSCs). Muscle differentiation was assessed through immunofluorescence staining with muscle markers, including Myosin heavy chain (MHC), Myogenin (MYOG), and Desmin. The metabolic activity of Creatine Kinase in C2C12 differentiated cells significantly increased compared to proliferated cells. Quantitative PCR analysis revealed a significant increase in Myosin Heavy Polypeptide 1 (MYH1) and MYOG expression compared to Day 0. These results highlight the application of decellularised plant scaffold (DPS) as a promising, edible material conducive to cell attachment, proliferation, and differentiation into muscle tissue. To create a CM prototype with biological mimicry, pADMSC-derived muscle and fat cells were also co-cultured on the same scaffold. The co-culture was confirmed through immunofluorescence staining of muscle markers and LipidTOX staining, revealing distinct muscle fibres and adipocytes containing lipid droplets respectively. Texture profile analysis conducted on uncooked CM prototypes and pork loin showed no significant differences in textural values. However, the pan-fried CM prototype differed significantly in hardness and chewiness compared to pork loin. Understanding the scaffolds’ textural profile enhances our insight into the potential sensory attributes of CM products. DPS shows potential for advancing CM biomanufacturing.

Comparison of skeletal muscle decellularization protocols and recellularization with adipose-derived stem cells for tissue engineering

Decellularization is a novel technique employed for scaffold manufacturing, as a strategy for skeletal muscle (SM) tissue engineering applications. However, poor decellularization efficacy is still a problem for the use of decellularized scaffolds as truly biocompatible biomaterials. For recellularization, adipose-derived stem cells (ASCs) are a good option, due to their immunomodulatory and pro-regenerative capacity, but few studies have described their combination with muscle-decellularized matrices (mDMs). This work aimed to evaluate the efficiency of four multi-step decellularization protocols to produce mDMs and to investigate in vitro biocompatibility with ASCs. Here, we described the different efficacies of muscle decellularization methods, suggesting the need for stricter standardization of the method, considering the large range of applications in SM tissue engineering, which is also a promising platform for preclinical studies with rat disease models using autologous cells.

Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling.

Stretchable Alginate/GelMA Interpenetrating Network (IPN) hydrogel microsprings based on coaxial microfluidic technique for skeletal muscle tissue engineering

The emulation of the natural organization and contractile properties of native stretchable tissues have crucial implications for skeletal muscle tissue engineering (SMTE). Helical structures, ubiquitous in nature and endowed with unique mechanical characteristics, have recently gained significant traction in SMTE applications. However, developing hierarchical hydrogel-based scaffolds that both replicate exceptional mechanical properties and control cellular organization remains challenging. Herein, we present an interpenetrating network (IPN) hydrogel synthesized from alginate and GelMA materials, to fabricate stretchable helical hydrogel microsprings via a coaxial microfluidic chip, showcasing superior elasticity attributed to helical structures. These hydrogel microsprings showed the micro- and nanoscale patterned groove/ridge helical structures, which microsprings demonstrated the ability to guide myoblast alignment and elongation along the longitudinal axis of the helical structures in vitro. Moreover, when the hydrogel microsprings were tailored into suitable sizes and utilized as injectable micro-scaffolds, they provided a conducive mechanical microenvironment for enhancing cell infiltration and facilitating muscle tissue regeneration in situ in rat models. This study offers a promising strategy for creating stretchable helical hydrogel microsprings that integrate distinctive micro- and nanoscale features, which not only showed the potential as biomimetic elastic scaffolds for guiding 3D cellular orientation in vitro but also as injectable carriers for enhancing skeletal muscle tissue regeneration in vivo.

Repairing Volumetric Muscle Loss with Commercially Available Hydrogels in an Ovine Model.

Volumetric muscle loss (VML) is the loss of skeletal muscle that exceeds the muscle's self-repair mechanism and leads to permanent functional deficits. In a previous study, we demonstrated the ability of our scaffold-free, multiphasic, tissue-engineered skeletal muscle units (SMUs) to restore muscle mass and force production. However, it was observed that the full recovery of muscle structure was inhibited due to increased fibrosis in the repair site. As such, novel biomaterials such as hydrogels (HGs) may have significant potential for decreasing the acute inflammation and subsequent fibrosis, as well as enhancing skeletal muscle regeneration following VML injury and repair. The goal of the current study was to assess the biocompatibility of commercially available poly(ethylene glycol), methacrylated gelatin, and hyaluronic acid (HA) HGs in combination with our SMUs to treat VML in a clinically relevant large animal model. An acute 30% VML injury created in the sheep peroneus tertius (PT) muscle was repaired with or without HGs and assessed for acute inflammation (incision swelling) and white blood cell counts in blood for 7 days. At the 7-day time point, HA was selected as the HG to use for the combined HG/SMU repair, as it exhibited a reduced inflammation response compared to the other HGs. Six weeks after implantation, all groups were assessed for gross and histological structural recovery. The results showed that the groups repaired with an SMU (SMU-Only and SMU+HA) restored muscle mass to greater degree than the groups with only HG and that the SMU groups had PT muscle masses that were statistically indistinguishable from its uninjured contralateral PT muscle. Furthermore, the HA HG, SMU-Only, and SMU+HA groups displayed notable efficacy in diminishing pro-inflammatory markers and showed an increased number of regenerating muscle fibers in the repair site. Taken together, the data demonstrates the efficacy of HA HG in decreasing acute inflammation and fibrotic response. The combination of HA and our SMUs also holds promise to decrease acute inflammation and fibrosis and increase muscle regeneration, advancing this combination therapy toward clinically relevant interventions for VML injuries in humans.

Trends and Future Research in Skeletal Muscle Tissue Engineering in the Past Decade (2012-2022).

To learn about advances in skeletal muscle tissue engineering (SMTE) in recent years, we used VOSviewer and Citespace software to quantitatively analyze and visualize relevant literature in the Web of Science database during the period 2012-2022. By mapping high-frequency keyword relationship networks, keyword time zones, and journal article cocitations, we clarified the areas of great interest, evolutionary paths, and developmental trends in research on SMTE. We conducted an in-depth analysis of highly cited and representative articles at various stages to summarize the mainstream research areas of great interest in SMTE and discussed the future development and challenges in this field, intending to provide a reference for the clinical treatment of skeletal muscle injury repair. We found that a collaborative network of authors has formed in this field; the journals publishing SMTE articles belong to the fields of biomaterials and tissue engineering, and open-access journals have played a key role in the promotion of the development of SMTE; and in the past decade, there has been rapid progress in SMTE research in terms of both depth and breadth. Impact statement Compared with the literature review method, bibliometrics can provide a comprehensive knowledge of a knowledge area based on a huge amount of literature. In this article, based on the Web of Science database, CiteSpace, and Vosviewer visualization tools were used to measure and analyze the literature reports in the field of skeletal muscle tissue engineering (SMTE). The research hotspots and cutting-edge information on SMTE were mined in terms of the number of publications, the number of citations, the keywords, the authors, and the publishing institutions to understand the current status of the research on SMTE in the world, to provide a reference for related researchers, engineering research in the field of SMTE, to comprehensively understand the current status of global research in the field of SMTE, and to provide a reference for related researchers.

Schwann Cells Do Not Promote Myogenic Differentiation in the EPI Loop Model.

In skeletal muscle tissue engineering, innervation and vascularization play an essential role in the establishment of functional skeletal muscle. For adequate three-dimensional assembly, biocompatible aligned nanofibers are beneficial as matrices for cell seeding. The aim of this study was to analyze the impact of Schwann cells (SC) on myoblast (Mb) and adipogenic mesenchymal stromal cell (ADSC) cocultures on poly-ɛ-caprolactone (PCL)-collagen I-nanofibers in vivo. Human Mb/ADSC cocultures, as well as Mb/ADSC/SC cocultures, were seeded onto PCL-collagen I-nanofiber scaffolds and implanted into the innervated arteriovenous loop model (EPI loop model) of immunodeficient rats for 4 weeks. Histological staining and gene expression were used to compare their capacity for vascularization, immunological response, myogenic differentiation, and innervation. After 4 weeks, both Mb/ADSC and Mb/ADSC/SC coculture systems showed similar amounts and distribution of vascularization, as well as immunological activity. Myogenic differentiation could be observed in both groups through histological staining (desmin, myosin heavy chain) and gene expression (MYOD, MYH3, ACTA1) without significant difference between groups. Expression of CHRNB and LAMB2 also implied neuromuscular junction formation. Our study suggests that the addition of SC did not significantly impact myogenesis and innervation in this model. The implanted motor nerve branch may have played a more significant role than the presence of SC.

DEVELOPMENT OF 4D PRINTING STRATEGY FOR SKELETAL MUSCLE TISSUE ENGINEERING

Skeletal muscle tissue engineering has made progress towards production of functional tissues in line with the development in materials science and fabrication techniques. In particular, combining the specificity of 3D printing with smart materials has introduced a new concept called the 4D printing. Inspired by the unique properties of smart/responsive materials, we designed a bioink made of gelatin, a polymer with well-known cell compatibility, to be 3D printed on a magnetically responsive substrate. Gelatin was made photocrosslinkable by the methacrylate reaction (GELMA), and its viscosity was finetuned by blending with alginate which was later removed by alginate lyase treatment, so that the printability of the bioink as well as the cell viability can be finetuned. C2C12 mouse myoblasts-laden bioink was then 3D printed on a magnetic substrate for 4D shape-shifting. The magnetic substrate was produced using silicon rubber (EcoFlex) and carbonyl iron powders. After 3D printing, the bioink was crosslinked on the substrate, and the substrate was rolled with the help of a permanent magnet. Unrolled (Open) samples were used as the control group. The stiffness of the bioink matrix was found to be in the range of 13–45 kPa, which is the appropriate value for the adhesion of C2C12 cells. In the cell viability analysis, it was observed that the cells survived and could proliferate within the 7-day duration of the experiment. As a result of the immunofluorescence test, compared to the Open Group, more cell nuclei were observed overlapping MyoD1 expression in the Rolled Group; this indicated that the cells in these samples had more cell-cell interactions and therefore tended to form more myotubes.Acknowledgements: This research was supported by the TÜBİTAK 2211-A and YÖK 100/2000 scholarship programs.

Hijacking plant skeletons for biomedical applications: from regenerative medicine and drug delivery to biosensing.

The field of biomedical engineering continually seeks innovative technologies to address complex healthcare challenges, ranging from tissue regeneration to drug delivery and biosensing. Plant skeletons offer promising opportunities for these applications due to their unique hierarchical structures, desirable porosity, inherent biocompatibility, and adjustable mechanical properties. This review comprehensively discusses chemical principles underlying the utilization of plant-based scaffolds in biomedical engineering. Highlighting their structural integrity, tunable properties, and possibility of chemical modification, the review explores diverse preparation strategies to tailor plant skeleton properties for bone, neural, cardiovascular, skeletal muscle, and tendon tissue engineering. Such applications stem from the cellulosic three-dimensional structure of different parts of plants, which can mimic the complexity of native tissues and extracellular matrices, providing an ideal environment for cell adhesion, proliferation, and differentiation. We also discuss the application of plant skeletons as carriers for drug delivery due to their structural diversity and versatility in encapsulating and releasing therapeutic agents with controlled kinetics. Furthermore, we present the emerging role played by plant-derived materials in biosensor development for diagnostic and monitoring purposes. Challenges and future directions in the field are also discussed, offering insights into the opportunities for future translation of sustainable plant-based technologies to address critical healthcare needs.

Assessment of laser-synthesized Si nanoparticle effects on myoblast motility, proliferation and differentiation: towards potential tissue engineering applications.

Due to their biocompatibility and biodegradability and their unique structural and physicochemical properties, laser-synthesized silicon nanoparticles (Si-NPs) are one of the nanomaterials which have been most studied as potential theragnostic tools for non-invasive therapeutic modalities. However, their ability to modulate cell behavior and to promote proliferation and differentiation is still very little investigated or unknown. In this work, ultrapure ligand free Si-NPs of 50 ± 11.5 nm were prepared by femtosecond (fs) laser ablation in liquid. After showing the ability of Si-NPs to be internalized by murine C2C12 myoblasts, the cytotoxicity of the Si-NPs on these cells was evaluated at concentrations ranging from 14 to 224 μg mL-1. Based on these findings, three concentrations of 14, 28 and 56 μg mL-1 were thus considered to study the effect on myoblast differentiation, proliferation and motility at the molecular and phenotypical levels. It was demonstrated that up to 28 μg mL-1, the Si-NPs are able to promote the proliferation of myoblasts and their subsequent differentiation. Scratch tests were also performed revealing the positive Si-NP effect on cellular motility at 14 and 28 μg mL-1. Finally, gene expression analysis confirmed the ability of Si-NPs to promote proliferation, differentiation and motility of myoblasts even at very low concentration. This work opens up novel exciting prospects for Si-NPs made by the laser process as innovative tools for skeletal muscle tissue engineering in view of developing novel therapeutic protocols for regenerative medicine.

Enhancement of mitochondrial energy metabolism by melatonin promotes vascularized skeletal muscle regeneration in a volumetric muscle loss model

Volumetric muscle loss (VML) is a condition that results in the extensive loss of 20 % or more of skeletal muscle due to trauma or tumor ablation, leading to severe functional impairment and permanent disability. The current surgical interventions have limited functional regeneration of skeletal muscle due to the compromised self-repair mechanism. Melatonin has been reported to protect skeletal muscle from exercise-induced oxidative damage and holds great potential to treat muscle diseases. In this study, we hypothesize that melatonin can enhance myoblast differentiation and promote effective recovery of skeletal muscle following VML. In vitro administration of melatonin resulted in a significant enhancement of myogenesis in C2C12 myoblast cells, as evidenced by the up-regulation of myogenic marker genes in a dose-dependent manner. Further experiments revealed that silent information of regulator type 3 (SIRT3) played a critical role in the melatonin-enhanced myoblast differentiation through enhancement of mitochondrial energy metabolism and activation of mitochondrial antioxidant enzymes such as superoxide dismutase 2 (SOD2). Silencing of Sirt3 completely abrogated the protective effect of melatonin on the mitochondrial function of myoblasts, evidenced by the increased reactive oxygen species, decreased adenosine triphosphate production, and down-regulated myoblast-specific marker gene expression. In order to attain a protracted and consistent release, liposome-encapsuled melatonin was integrated into gelatin methacryloyl hydrogel (GelMA-Lipo@MT). The implantation of GelMA-Lipo@MT into a tibialis anterior muscle defect in a VML model effectively stimulated the formation of myofibers and new blood vessels in situ, while concurrently inhibiting fibrotic collagen deposition. The findings of this study indicate that the incorporation of melatonin with GelMA hydrogel has facilitated the de novo vascularized skeletal muscle regeneration by augmenting mitochondrial energy metabolism. This represents a promising approach for the development of skeletal muscle tissue engineering, which could be utilized for the treatment of VML and other severe muscle injuries.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.