Regenerative Capacity Of Skeletal Muscle Research Articles

Mitochondrial Dynamics Drive Muscle Stem Cell Progression from Quiescence to Myogenic Differentiation.

From quiescence to activation and myogenic differentiation, muscle stem cells (MuSCs) experience drastic alterations in their signaling activity and metabolism. Through balanced cycles of fission and fusion, mitochondria alter their morphology and metabolism, allowing them to affect their decisive role in modulating MuSC activity and fate decisions. This tightly regulated process contributes to MuSC regulation by mediating changes in redox signaling pathways, cell cycle progression, and cell fate decisions. In this review, we discuss the role of mitochondrial dynamics as an integral modulator of MuSC activity, fate, and maintenance. Understanding the influence of mitochondrial dynamics in MuSCs in health and disease will further the development of therapeutics that support MuSC integrity and thus may aid in restoring the regenerative capacity of skeletal muscle.

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.

Novel muscle-derived extracellular matrix hydrogel promotes angiogenesis and neurogenesis in volumetric muscle loss

Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical properties and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model.

NEDD4-1 deficiency impairs satellite cell function during skeletal muscle regeneration

BackgroundSatellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin–proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined.ResultsUsing conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter.ConclusionsThese results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.

Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration.

Age-related decline in skeletal muscle regenerative capacity is multifactorial, yet the contribution of immune dysfunction to regenerative failure is unknown. Macrophages are essential for effective debris clearance and muscle stem cell activity during muscle regeneration, but the regulatory mechanisms governing macrophage function during muscle repair are largely unexplored. Here, we uncover a new mechanism of immune modulation operating during skeletal muscle regeneration that is disrupted in aged animals and relies on the regulation of macrophage function. The immune modulator mesencephalic astrocyte-derived neurotrophic factor (MANF) is induced following muscle injury in young mice but not in aged animals, and its expression is essential for regenerative success. Regenerative impairments in aged muscle are associated with defects in the repair-associated myeloid response similar to those found in MANF-deficient models and could be improved through MANF delivery. We propose that restoring MANF levels is a viable strategy to improve myeloid response and regenerative capacity in aged muscle.

Loss of CRY2 promotes regenerative myogenesis by enhancing PAX7 expression and satellite cell proliferation.

The regenerative capacity of skeletal muscle is dependent on satellite cells. The circadian clock regulates the maintenance and function of satellite cells. Cryptochrome 2 (CRY2) is a critical component of the circadian clock, and its role in skeletal muscle regeneration remains controversial. Using the skeletal muscle lineage and satellite cell-specific CRY2 knockout mice (CRY2scko), we show that the deletion of CRY2 enhances muscle regeneration. Single myofiber analysis revealed that deletion of CRY2 stimulates the proliferation of myoblasts. The differentiation potential of myoblasts was enhanced by the loss of CRY2 evidenced by increased expression of myosin heavy chain (MyHC) and myotube formation in CRY2-/- cells versus CRY2+/+ cells. Immunostaining revealed that the number of mononucleated paired box protein 7 (PAX7+) cells associated with myotubes formed by CRY2-/- cells was increased compared with CRY2+/+ cells, suggesting that more reserve cells were produced in the absence of CRY2. Loss of CRY2 leads to the activation of the ERK1/2 signaling pathway and ETS1, which binds to the promoter of PAX7 to induce its transcription. CRY2 deficient myoblasts survived better in ischemic muscle. Therefore, CRY2 is essential in regulating skeletal muscle repair.

A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Injectable conductive micro-cryogel as a muscle stem cell carrier improves myogenic proliferation, differentiation and in situ skeletal muscle regeneration

Volumetric muscle loss (VML) results in the impediment of skeletal muscle function, and there were still great challenges in cell delivery approach with the minimally invasive operation to repair muscle defects. To deliver cells to the VML defects site efficiently, the injectable conductive porous nanocomposite microcryogels based on gelatin (GT) and reduced graphene oxide (rGO) were designed and prepared. The microcryogels were loaded with myoblasts to form an injectable cell delivery system and show the ability to protect cells during injection. Conductive microcryogel with 4 mg/mL rGO (GT/rGO4) enhanced C2C12 cell proliferation and myogenic differentiation during 3D culture compared with pure gelatin microcryogel. In a mice VML model, injection of microcryogel loaded with muscle-derived stem cells into the injury site significantly improved the generation of new muscle fibers and blood vessels, and anti-inflammatory properties. The results show that injectable biodegradable conductive microcryogel can be used as muscle stem cell carriers with the potential to maintain cell activity and deliver cells to defective sites, thereby in situ enhancing skeletal muscle regeneration. Statement of significanceVolumetric muscle loss overwhelms the regenerative capacity of skeletal muscle, which results in severe damage to muscle tissues. In the treatment of volumetric muscle loss, conductive niche and muscle stem cells are needed to alleviate excessive scar formation and inflammation to improve muscle regeneration. Injectable gelatin/reduced graphene oxide based nanocomposite microcryogel can enhance the differentiation of seeded muscle stem cells. The improved repair of volumetric muscle loss was achieved via reducing collagen deposition and inflammation in the injected region through the microcryogel cell-delivery therapy, suggesting great potential of the injectable microcryogel as a cell carrier in soft tissue repair.

Ethanol Shifts Myoblast Bioenergetic Phenotype: Implications for Muscle Regeneration

At‐risk alcohol use is nearly twice as prevalent among people living with HIV (PLWH) as the general population. Alcohol and HIV can independently and synergistically increase risk for metabolic diseases, including myopathy. Decreased functional skeletal muscle (SKM) mass is associated with morbidity and all‐cause mortality. A major focus of our research is to investigate the independent and combined effects of alcohol and HIV on SKM regenerative capacity and its contribution to myopathy development. Previously, we showed that in vitro ethanol decreases myoblast extracellular acidification rate (ECAR), indicative of decreased glycolytic function, and that the decrease in ECAR was associated with decreased myoblast differentiation. The objective of this study was to determine effects of prior and recent alcohol exposure and simian immunodeficiency virus (SIV) on bioenergetic function in myoblasts from naïve and SIV‐infected rhesus macaques. Female rhesus macaques (6‐10 years of age) were administered chronic binge alcohol (CBA; peak blood alcohol concentration=50‐60 mM, 5 days per week) or isovolumetric water vehicle (VEH) beginning 3 months prior to infection with SIVmac251. Daily antiretroviral therapy (ART) was initiated 2.5 months after SIV infection, and macaques were humanely euthanized 9 months later. SKM samples were collected for myoblast isolation prior to CBA or VEH (naïve, N=6) and at study end point (VEH/SIV, N=6; CBA/SIV, N=6). Myoblasts from each group were incubated with ethanol (0 and 50 mM) for 3 days prior to Mito Stress Test, Glycolysis Stress Test, and ATP Rate Assay using an XFe96 Extracellular Flux Analyzer. There were no significant differences due to CBA or SIV on mitochondrial or glycolytic function for any measure. During the Mito Stress Test, ethanol exposure significantly (p<0.05) decreased ECAR at baseline and after the addition of oligomycin (ATP synthase inhibitor), indicative of decreased glycolytic function, without significant changes in mitochondrial function. Similarly, ethanol exposure decreased all parameters in the Glycolysis Stress Test (non‐glycolytic acidification, glycolysis, glycolytic reserve, and glycolytic capacity). An ATP rate assay confirmed that ethanol exposure significantly decreased glycolytic ATP production, contributing to a decrease in total ATP production. These results indicate that recent exposure to an intoxicating concentration of ethanol shifts myoblast bioenergetic phenotype, impairs glycolysis, and produces a bioenergetic deficit regardless of prior alcohol exposure or SIV/ART. The shift away from glycolysis occurs under basal and stressed conditions. We propose that altered glycolytic enzyme activity underlies the alcohol‐mediated decrease in glycolytic function, negatively impacting myoblast differentiation and SKM regenerative capacity.

Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states.

Muscle stem cells (MuSCs) are essential for tissue homeostasis and regeneration, but the potential contribution of MuSC morphology to in vivo function remains unknown. Here, we demonstrate that quiescent MuSCs are morphologically heterogeneous and exhibit different patterns of cellular protrusions. We classified quiescent MuSCs into three functionally distinct stem cell states: responsive, intermediate, and sensory. We demonstrate that the shift between different stem cell states promotes regeneration and is regulated by the sensing protein Piezo1. Pharmacological activation of Piezo1 is sufficient to prime MuSCs toward more responsive cells. Piezo1 deletion in MuSCs shifts the distribution toward less responsive cells, mimicking the disease phenotype we find in dystrophic muscles. We further demonstrate that Piezo1 reactivation ameliorates the MuSC morphological and regenerative defects of dystrophic muscles. These findings advance our fundamental understanding of how stem cells respond to injury and identify Piezo1 as a key regulator for adjusting stem cell states essential for regeneration.

Laminin-111-Enriched Fibrin Hydrogels Enhance Functional Muscle Regeneration Following Trauma.

Volumetric muscle loss (VML) is the surgical or traumatic loss of skeletal muscle, which can cause loss of limb function or permanent disability. VML injuries overwhelms the endogenous regenerative capacity of skeletal muscle and results in poor functional healing outcomes. Currently, there are no approved tissue engineering treatments for VML injuries. In this study, fibrin hydrogels enriched with laminin-111 (LM-111; 50-450 μg/mL) were used for the treatment of VML of the tibialis anterior in a rat model. Treatment with fibrin hydrogel containing 450 μg/mL of LM-111 (FBN450) improved muscle regeneration following VML injury. FBN450 hydrogel treatment increased the relative proportion of contractile to fibrotic tissue as indicated by the myosin: collagen ratio on day 28 post-VML injury. FBN450 hydrogels also enhanced myogenic protein expression and increased the quantity of small to medium size myofibers (500-2000 μm2) as well as innervated myofibers. Improved contractile tissue deposition due to FBN450 hydrogel treatment resulted in a significant improvement (∼60%) in torque production at day 28 postinjury. Taken together, these results suggest that the acellular FBN450 hydrogels provide a promising therapeutic strategy for VML that is worthy of further investigation. Impact statement Muscle trauma accounts for 50-70% of total military injuries and complications involving muscle result in ∼80% of delayed amputations. The lack of a clinical standard of care for volumetric muscle loss (VML) injuries presents an opportunity to develop novel regenerative therapies and improve healing outcomes. Laminin-111-enriched fibrin hydrogel may provide a promising therapy for VML that is worthy of further investigation. The acellular nature of these hydrogels will allow for easy off the shelf access to critically injured patients and fewer regulatory hurdles during commercialization.

Unresolved intramuscular inflammation, not diminished skeletal muscle regenerative capacity, is at the root of rheumatoid cachexia: insights from a rat CIA model

Rheumatoid arthritis targets numerous organs in patients, including the skeletal muscle, resulting in rheumatoid cachexia. In the muscle niche, satellite cells, macrophages, and myofibroblasts may be affected and the factors they release altered. This study aimed to assess these cell types, cytokines, and growth factors and their relationships to muscle fiber size and number in a rodent collagen‐induced arthritis (CIA) model, in order to identify new therapeutic targets. Fiber cross‐sectional area (CSA) was 57% lower in CIA than controls (p < 0.0001), thus smaller but more fibers visible per field of view. Immunostaining indicated the increased presence of satellite cells, macrophages, myofibroblasts, and myonuclei per field of view in CIA (p < 0.01), but this finding was not maintained when taking fiber number into consideration. Western blots of gastrocnemius samples indicated that tumor necrosis factor‐α was significantly elevated (p < 0.01) while interleukin‐10 (IL‐10) was decreased (p < 0.05) in CIA. This effect was maintained (and heightened for IL‐10) when expressed per fiber number. Myogenic regulatory factors (MyoD and myogenin), transforming growth factor‐β and inhibitor of differentiation were significantly elevated in CIA muscle and levels correlated significantly with CSA. Several of these factors remained elevated, but bone morphogenetic protein‐7 decreased when considering fiber number per area. In conclusion, CIA‐muscle demonstrated a good regenerative response. Myoblast numbers per fiber were not elevated, suggesting their activity results from the persistent inflammatory signaling which also significantly hampered maintenance of muscle fiber size. A clearer picture of signaling events at cellular level in arthritis muscle may be derived from expressing data per fiber.

QS6: Collagen-glycosaminoglycans Scaffold Can Improve Muscle Function Recovery After Volumetric Muscle Loss Injury in Rat Model

Propose:Skeletal muscle regenerative capacity fails in restoring muscle function following major injuries in which significant skeletal muscle is lost or damaged. As such, volumetric muscle loss (VML), ultimately results in permanent disability. Therapeutic options are limited in VML and currently no standard of care exists. Biodegradable scaffolds hold great potentials in the regeneration of lost muscle tissue by replacing the lost structural framework of the defect. In this study we aim to evaluate the effects of a porous collagen-glycosaminoglycans scaffold (CGS) on muscle function recovery following VML injury in a rat model.Methods:Fifteen male 12-week old Sprague-Dawley rats were randomly divided into three groups of 5 rats in each: 1) Sham group, 2) VML Untreated (control) group, and 3) VML+CGS group. A standard model of VML injury was performed to bilateral Tibialis Anterior (TA) muscles in VML Untreated and VML+CGS groups. The muscle defect in VML+CGS group was filled with 3 sheets of CGS cuts while the muscle defects in VML control group left untreated. Animals in Sham group underwent the same surgical procedure (skin and fascia opening and closure) but without any muscle injury. Pick isometric twitch and tetanus forces of foot dorsiflexion were measured in vivo prior, immediately after, and four weeks post injury using a dual mode muscle lever system. In situ TA muscle twitch and tetanic contraction strengths were also determined six weeks after injury.Results:Prior, and immediately after injury no statistically significant differences was seen between VML Untreated and VML+CGS groups in terms of the means of twitch and tetanus foot isometric dorsiflexion forces. Four weeks after injury, TA of VML+ CGS group showed significant functional recovery as compared with VML Untreated group in twitch (76 ± 22.7% vs 51.3 ± 22.3%, p<0.01) and tetanus (77.4 ± 22.1% vs 59.8 ± 22.4%, p<0.05) foot dorsiflexion forces. In situ muscle strength measurements of TA at six weeks post injury, also showed significantly higher twitch (35.2 ± 12.5 mN/mm2 vs 23.7 ± 6.9 mN/mm2, p<0.05) and tetanus (115.0 ± 35.3 mN/mm2 vs 87.9 ± 20 mN/mm2, p<0.05) forces in TA of VML+CGS group when compared with the VML Untreated group. On histology of the TA performed six weeks following initial injury, placement of CGS decreased fibrosis across the injury, as defined as percentage of positive area in trichrome staining compared with VML Untreated group (1.3 ± 0.1% vs. 7.1 ± 1.3%, p<0.01). In addition, immunofluorescence tissue staining confirmed positive myosin heavy chain staining within the scaffold, indicating the formation of nascent myofibers within the area of injury.Conclusions:Application of CGS to the muscle defect following VML injury can improve muscle function recovery, limit fibrosis, and promote muscle regeneration in rodent model. These findings can foster future research in larger animals and human subjects.*p<0.05, **p<0.01. Error bars: Standard Deviations.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Skeletal Muscle Regeneration Impairment in Type 1 Diabetes Mellitus is Characterized by Dysregulated Sphingosine‐1‐Phosphate and Ceramide‐1‐Phosphate Responses

Type 1 diabetes mellitus (T1DM) impairs regenerative capacity of skeletal muscle, one of several aspects of diabetic myopathy. It was hypothesized that accumulation of lipid species within the muscle (lipotoxicity) could cause this impairment. Although it has been demonstrated that diabetes causes increased lipid deposition in skeletal muscle, the muscle lipid response following tissue damage has not been investigated. To assess the effect of T1DM on lipid deposition in regenerating skeletal muscle, wild-type (WT) and T1DM (Akita) mice (n=5) received cardiotoxin (CTX) injections into the left tibialis anterior (TA). TAs were collected at 5-days post-CTX and subsequently cryosectioned and stained with BODIPY 493/503 to visualize lipids. Microscope images were analyzed for proportion of area positive for lipid via thresholding. Injured Akita muscle was found to have more total lipid as compared to uninjured (control) Akita, and both injured and control WT muscle (p<0.05). Next, we assessed individual lipid species via liquid chromatography-mass spectrometry across a broader time-course of muscle regeneration. Here, 22 WT and 21 Akita mice received CTX injections into the quadriceps which were harvested and frozen at 1, 3, 5, and 7 days post-CTX (n=4-6). 45 lipid species were analyzed via liquid chromatography-mass spectrometry, including sphingosine-1-phosphate, a critical sphingolipid for stimulating satellite cell activity. Injured quadriceps were found to have significantly elevated concentrations of S1P as compared to control particularly at 5 days post-CTX (p<0.05), however this response was blunted in the Akita quadriceps. No differences were found between groups in concentration of the precursors of S1P (d18:1), sphingomyelin (d18:1/12:0; d18:1/14:0; d18:1/16:0; d18:1/17:0; d18:1/18:0), sphinganine (d18:0; d18:1; d18:2; d20:0), dihydroceramide (d18:0/16:0; d18:0/18:1 9Z; d18:0/24:0; d18:0/22:0; d18:0/22:6), ceramide (d18:1/16:0; d18:1/18:1; d18:1/20:0; d18:1/22:0; d18:1/24:0; d18:1/24:1) and sphingosine (d18:1), suggesting that low S1P in regenerating Akita muscle could be due to dysregulation of its regulating enzymes, sphingosine kinase (SpK1) and sphingosine lyase (SL). Elevated SL expression was found in Akita injured muscle (p<0.05) while no changes were found in SpK1 expression or associated ERK1/2 signalling, suggesting that S1P breakdown is increased in T1DM. Ceramide-1-phosphate (C1P; d18:1, 16:0) was significantly elevated in Akita compared to WT muscle, peaking at 5 days post-CTX (p<0.05). The role of C1P in muscle regeneration is unknown. Taken together, these data indicate that at early time points, muscle regeneration is impaired in T1DM due to accelerated S1P breakdown while other lipid species such as C1P may play a role.

Ionic Silicon Protects Oxidative Damage and Promotes Skeletal Muscle Cell Regeneration.

Volumetric muscle loss injuries overwhelm the endogenous regenerative capacity of skeletal muscle, and the associated oxidative damage can delay regeneration and prolong recovery. This study aimed to investigate the effect of silicon-ions on C2C12 skeletal muscle cells under normal and excessive oxidative stress conditions to gain insights into its role on myogenesis during the early stages of muscle regeneration. In vitro studies indicated that 0.1 mM Si-ions into cell culture media significantly increased cell viability, proliferation, migration, and myotube formation compared to control. Additionally, MyoG, MyoD, Neurturin, and GABA expression were significantly increased with addition of 0.1, 0.5, and 1.0 mM of Si-ion for 1 and 5 days of C2C12 myoblast differentiation. Furthermore, 0.1–2.0 mM Si-ions attenuated the toxic effects of H2O2 within 24 h resulting in increased cell viability and differentiation. Addition of 1.0 mM of Si-ions significantly aid cell recovery and protected from the toxic effect of 0.4 mM H2O2 on cell migration. These results suggest that ionic silicon may have a potential effect in unfavorable situations where reactive oxygen species is predominant affecting cell viability, proliferation, migration, and differentiation. Furthermore, this study provides a guide for designing Si-containing biomaterials with desirable Si-ion release for skeletal muscle regeneration.

Aging of the immune system and impaired muscle regeneration: A failure of immunomodulation of adult myogenesis.

Skeletal muscle regeneration that follows acute injury is strongly influenced by interactions with immune cells that invade and proliferate in the damaged tissue. Discoveries over the past 20years have identified many of the key mechanisms through which myeloid cells, especially macrophages, regulate muscle regeneration. In addition, lymphoid cells that include CD8+ T-cells and regulatory T-cells also significantly affect the course of muscle regeneration. During aging, the regenerative capacity of skeletal muscle declines, which can contribute to progressive loss of muscle mass and function. Those age-related reductions in muscle regeneration are accompanied by systemic, age-related changes in the immune system, that affect many of the myeloid and lymphoid cell populations that can influence muscle regeneration. In this review, we present recent discoveries that indicate that aging of the immune system contributes to the diminished regenerative capacity of aging muscle. Intrinsic, age-related changes in immune cells modify their expression of factors that affect the function of a population of muscle stem cells, called satellite cells, that are necessary for normal muscle regeneration. For example, age-related reductions in the expression of growth differentiation factor-3 (GDF3) or CXCL10 by macrophages negatively affect adult myogenesis, by disrupting regulatory interactions between macrophages and satellite cells. Those changes contribute to a reduction in the numbers and myogenic capacity of satellite cells in old muscle, which reduces their ability to restore damaged muscle. In addition, aging produces changes in the expression of molecules that regulate the inflammatory response to injured muscle, which also contributes to age-related defects in muscle regeneration. For example, age-related increases in the production of osteopontin by macrophages disrupts the normal inflammatory response to muscle injury, resulting in regenerative defects. These nascent findings represent the beginning of a newly-developing field of investigation into mechanisms through which aging of the immune system affects muscle regeneration.

3D Printing Decellularized Extracellular Matrix to Design Biomimetic Scaffolds for Skeletal Muscle Tissue Engineering

Functional engineered muscles are still a critical clinical issue to be addressed, although different strategies have been considered so far for the treatment of severe muscular injuries. Indeed, the regenerative capacity of skeletal muscle (SM) results inadequate for large-scale defects, and currently, SM reconstruction remains a complex and unsolved task. For this aim, tissue engineered muscles should provide a proper biomimetic extracellular matrix (ECM) alternative, characterized by an aligned/microtopographical structure and a myogenic microenvironment, in order to promote muscle regeneration. As a consequence, both materials and fabrication techniques play a key role to plan an effective therapeutic approach. Tissue-specific decellularized ECM (dECM) seems to be one of the most promising material to support muscle regeneration and repair. 3D printing technologies, on the other side, enable the fabrication of scaffolds with a fine and detailed microarchitecture and patient-specific implants with high structural complexity. To identify innovative biomimetic solutions to develop engineered muscular constructs for the treatment of SM loss, the more recent (last 5 years) reports focused on SM dECM-based scaffolds and 3D printing technologies for SM regeneration are herein reviewed. Possible design inputs for 3D printed SM dECM-based scaffolds for muscular regeneration are also suggested.

Synergistic effect of high-intensity interval training and stem cell transplantation with amniotic membrane scaffold on repair and rehabilitation after volumetric muscle loss injury.

Despite the high regenerative capacity of skeletal muscle, volumetric muscle loss (VML) is an irrecoverable injury. One therapeutic approach is the implantation of engineered biologic scaffolds enriched with stem cells. The objective of this study is to investigate the synergistic effect of high-intensity interval training (HIIT) and stem cell transplantation with an amniotic membrane scaffold on innervation, vascularization and muscle function after VML injury. A VML injury was surgically created in the tibialis anterior (TA) muscle in rats. The animals were randomly assigned to three groups: untreated negative control group (untreated), decellularized human amniotic membrane bio-scaffold group (dHAM) and dHAM seeded with adipose-derived stem cells, which differentiate into skeletal muscle cells (dHAM-ADSCs). Then, each group was divided into sedentary and HIIT subgroups. The exercise training protocol consisted of treadmill running for 8weeks. The animals underwent in vivo functional muscle tests to evaluate maximal isometric contractile force. Regenerated TA muscles were harvested for molecular analyses and explanted tissues were analyzed with histological methods. The main finding was that HIIT promoted muscle regeneration, innervation and vascularization in regenerated areas in HIIT treatment subgroups, especially in the dHAM-ADSC subgroup. In parallel with innervation, maximal isometric force also increased in vivo. HIIT upregulated neurotrophic factor gene expression in skeletal muscle. The amniotic membrane bio-scaffold seeded with differentiated ADSC, in conjunction with exercise training, improved vascular perfusion and innervation and enhanced the functional and morphological healing process after VML injury. The implications of these findings are of potential importance for future efforts to develop engineered biological scaffolds and for the use of interval training programs in rehabilitation after VML injury.

Impaired ECM Remodeling and Macrophage Activity Define Necrosis and Regeneration Following Damage in Aged Skeletal Muscle.

Regenerative capacity of skeletal muscle declines with age, the cause of which remains largely unknown. We investigated extracellular matrix (ECM) proteins and their regulators during early regeneration timepoints to define a link between aberrant ECM remodeling, and impaired aged muscle regeneration. The regeneration process was compared in young (three month old) and aged (18 month old) C56BL/6J mice at 3, 5, and 7 days following cardiotoxin-induced damage to the tibialis anterior muscle. Immunohistochemical analyses were performed to assess regenerative capacity, ECM remodeling, and the macrophage response in relation to plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), and ECM protein expression. The regeneration process was impaired in aged muscle. Greater intracellular and extramyocellular PAI-1 expression was found in aged muscle. Collagen I was found to accumulate in necrotic regions, while macrophage infiltration was delayed in regenerating regions of aged muscle. Young muscle expressed higher levels of MMP-9 early in the regeneration process that primarily colocalized with macrophages, but this expression was reduced in aged muscle. Our results indicate that ECM remodeling is impaired at early time points following muscle damage, likely a result of elevated expression of the major inhibitor of ECM breakdown, PAI-1, and consequent suppression of the macrophage, MMP-9, and myogenic responses.