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Abstract Titanium alloys represent the prevailing material employed in orthopedic implants, which are present in millions of patients worldwide. The prolonged presence of these implants in the human body has raised concerns about possible health effects. This study presents a comprehensive analysis of titanium implants and surrounding tissue samples obtained from patients who underwent revision surgery for therapeutic reasons. The surface of the implants exhibited nano-scale corrosion defects, and nanoparticles were deposited in adjacent samples. In addition, muscle in close proximity to the implant showed clear evidence of fibrotic proliferation, with titanium content in the muscle tissue increasing the closer it was to the implant. Transcriptomics analysis revealed SNAI2 upregulation and activation of PI3K/AKT signaling. In vivo rodent and zebrafish models validated that titanium implant or nanoparticles exposure provoked collagen deposition and disorganized muscle structure. Snai2 knockdown significantly reduced implant-associated fibrosis in both rodent and zebrafish models. Cellular experiments demonstrated that titanium dioxide nanoparticles (TiO2 NPs) induced fibrotic gene expression at sub-cytotoxic doses, whereas Snai2 knockdown significantly reduced TiO2 NPs-induced fibrotic gene expression. The in vivo and in vitro experiments collectively demonstrated that Snai2 plays a pivotal role in mediating titanium-induced fibrosis. Overall, these findings indicate a significant release of titanium nanoparticles from the implants into the surrounding tissues, resulting in muscular fibrosis, partially through Snai2-dependent signaling. Graphical Abstract

Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age‐related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine‐Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age‐related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients. [ABSTRACT FROM AUTHOR]

Objective: To determine whether acupotomy ameliorates immobilization-induced muscle contracture and fibrosis via Wnt/β-catenin signaling pathway. Methods: Thirty Wistar rats were randomly divided into 5 groups (n=6) by a random number table, including control, immobilization, passive stretching, acupotomy, and acupotomy 3 weeks (3-w) groups. The rat model of gastrocnemius contracture was established by immobilizing the right hind limb in plantar flexion for 4 weeks. Rats in the passive stretching group received passive stretching at gastrocnemius, a daily series of 10 repetitions for 30 s each at 30-s intervals for 10 consecutive days. Rats in the acupotomy and acupotomy 3-w groups received acupotomy once and combined with passive stretching at gastrocnemius a daily series of 10 repetitions for 30 s each at 30-s intervals for 10 consecutive days. Additionally, rats in the acupotomy 3-w group were allowed to walk freely for 3 weeks after 10-day therapy. After treatment, range of motion (ROM), gait analysis [i.e., paw area, stance/swing and maximum ratio of paw area to paw area duration (Max dA/dT)], gastrocnemius wet weight and the ratio of muscle wet weight to body weight (MWW/BW) were tested. Gastrocnemius morphometric and muscle fiber cross-sectional area (CSA) were assessed by hematoxylin-eosin staining. Fibrosis-related mRNA expressions (i.e., Wnt 1, β-catenin, axin-2, α-smooth muscle actin, fibronectin, and types I and III collagen) were measured using real-time quantitative polymerase chain reactions. Wnt 1, β-catenin and fibronectin concentrations were measured by enzyme-linked immunosorbent assay. Types I and III collagen in the perimysium and endomysium were analyzed using immunofluorescence. Results: Compared with the control group, ROM, gait function, muscle weight, MWW/BW and CSA were significantly decreased in the immobilization group (all P<0.01), while protein levels of types I and III collagen, Wnt 1, β-catenin, fibronectin and mRNA levels of fibrosis-related genes were obviously increased (all P<0.01). Treatment with passive stretching or acupotomy restored ROM and gait function and increased muscle wet weight, MWW/BW and CSA (all P<0.05), while protein expression levels of Wnt 1, β-catenin, fibronectin, types I and III collagen and mRNA levels of fibrosis-related genes were remarkably declined compared with the immobilization group (all P<0.05). Compared with passive stretching group, ROM, gait function, MWW was remarkably restored (all P<0.05), and mRNA levels of fibrosis-related genes as well as protein expression levels of Wnt 1, β-catenin, fibronectin, types I and III collagen in the acupotomy group were obviously decreased (all P<0.05). Compared with the acupotomy group, ROM, paw area, Max dA/dT, and MWW were restored (all P<0.05), and mRNA levels of fibrosis-related genes along with protein levels of Wnt 1, β-catenin, fibronectin, types I and III collagen in the acupotomy 3-w group were decreased (P<0.05). Conclusion: Improvements in motor function, muscle contractures, and muscle fibrosis induced by acupotomy correlates with the inhibition of Wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Botulinum Neurotoxin Type A (BoNT-A) injections using Ultrasound (US) guidance have led to research evaluating changes in muscle architecture. Controversy remains as to what constitutes increased Echo-Intensity (EI) in spastic muscles and whether this may affect outcomes. We aim to provide a narrative review of US muscle architecture changes following Central Nervous System (CNS) lesions and explore their relationship to spasticity. Medline, CINAHL, and Embase databases were searched with keywords: ultrasonography, hypertonia, spasticity, fibrosis, and Heckmatt. Three physicians reviewed the results of the search to select relevant papers. Reviews identified in the search were used as a resource to identify additional studies. A total of 68 papers were included. Four themes were identified, including histopathological changes in spastic muscle, effects of BoNT-A on the muscle structure, available US modalities to assess the muscle, and utility of US assessment in clinical spasticity. Histopathological studies revealed atrophic and fibrofatty changes after CNS lesions. Several papers described BoNT-A injections contributing to those modifications. These changes translated to increased EI. The exact significance of increased muscle EI remains unclear. The Modified Heckmatt Scale (MHS) is a validated tool for grading muscle EI in spasticity. The use of the US may be an important tool to assess muscle architecture changes in spasticity and improve spasticity management. Treatment algorithms may be developed based on the degree of EI. Further research is needed to determine the incidence and impact of these EI changes in spastic muscles. [ABSTRACT FROM AUTHOR]

Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and ultrastructural changes of muscle fibers in sarcopenia, mitochondria and their quality control pathways play relevant roles. Mononucleated muscle stem cells/satellite cells (MSCs) have been attributed a critical role in muscle repair after an injury. The involvement of mitochondria in supporting MSC-directed muscle repair is unclear. There is evidence that a reduction in mitochondrial biogenesis blunts muscle repair, thus indicating that the delivery of functional mitochondria to injured muscles can be harnessed to limit muscle fibrosis and enhance restoration of muscle function. Injection of autologous respiration-competent mitochondria from uninjured sites to damaged tissue has been shown to reduce infarct size and enhance cell survival in preclinical models of ischemia–reperfusion. Furthermore, the incorporation of donor mitochondria into MSCs enhances lung and cardiac tissue repair. This strategy has also been tested for regeneration purposes in traumatic muscle injuries. Indeed, the systemic delivery of mitochondria promotes muscle regeneration and restores muscle mass and function while reducing fibrosis during recovery after an injury. In this review, we discuss the contribution of altered MSC function to sarcopenia and illustrate the prospect of harnessing mitochondrial delivery and restoration of MSCs as a therapeutic strategy against age-related sarcopenia. [ABSTRACT FROM AUTHOR]

Skeletal muscle atrophy coincides with extensive fibrous tissue hyperplasia in muscle‐atrophied patients, and fibrous tissue plays a vital role in skeletal muscle function and hinders muscle fiber regeneration. However, effective drugs to manage skeletal muscle atrophy and fibrosis remain elusive. This study isolated and characterized exosomes derived from skeletal muscle satellite cells (MuSC‐Exo). The study investigated their effects on denervated skeletal muscle atrophy and fibrosis in Sprague Dawley (SD) rats via intramuscular injection. MuSC‐Exo demonstrated the potential to alleviate skeletal muscle atrophy and fibrosis. The underlying mechanism using single‐cell RNA sequencing data and functional analysis are analyzed. Mechanistic studies reveal close associations between fibroblasts and myoblasts, with the transforming growth factor β1 (TGF‐β1)‐Smad3‐Pax7 axis governing fibroblast activation in atrophic skeletal muscle. MuSC‐Exo intervention inhibited the TGF‐β1/Smad3 pathway and improved muscle atrophy and fibrosis. In conclusion, MuSC‐Exo‐based therapy may represent a novel strategy to alleviate skeletal muscle atrophy and reduce excessive fibrotic tissue by targeting Pax7 through the TGF‐β1/Smad3 pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: Quadriceps contracture is a condition that can be observed in young dogs, such as a congenital disability or complication following a hindlimb fracture. Although surgical treatments have been reported, it is difficult to restore normal limb function when the disease is advanced in stage. Rehabilitation early in the course of the disease may effectively improve progress. Using a rat model, we investigated the effectiveness of therapeutic ultrasound and neuromuscular electrical stimulation (NMES) for quadriceps contracture treatment. Quadriceps contracture was induced by immobilizing both hindlimbs for 14 days. Then, we compared the effectiveness of four rehabilitation programs, including spontaneous recovery, therapeutic ultrasound, NMES, and a therapeutic ultrasound and NMES combination on quadriceps contracture for 28 days. After completing 28 days of rehabilitation, the range of joint motion (ROM), muscle histopathology, and levels of fibrosis and anti-fibrosis-associated mRNA expression were measured. The results showed that the therapeutic ultrasound and NMES combination inhibited sarcomere length shortening, muscle atrophy, and muscle fibrosis in quadriceps contracture. Quadriceps contracture is a condition where the muscle–tendon unit is abnormally shortened. The treatment prognosis is guarded to poor depending on the progress of the disease. To improve the prognosis, we investigated the effectiveness of therapeutic ultrasound and NMES in treating quadriceps contracture in an immobilized rat model. Thirty-six Wistar rats were randomized into control, immobilization alone, immobilization and spontaneous recovery, immobilization and therapeutic ultrasound, immobilization and NMES, and immobilization and therapeutic ultrasound and NMES combination groups. The continuous therapeutic ultrasound (frequency, 3 MHz, intensity 1 W/cm2) and NMES (TENS mode, frequency 50 Hz; intensity 5.0 ± 0.8 mA) were performed on the quadriceps muscle. On Day 15, immobilization-induced quadriceps contracture resulted in a decreased ROM of the stifle joint, reduction in the sarcomere length, muscle atrophy, and muscle fibrosis. On Day 43, therapeutic ultrasound, NMES, and combining both methods improved muscle atrophy and shortening and decreased collagen type I and III and α-SMA protein. The combination of therapeutic ultrasound and NMES significantly reduced the mRNA expression of IL-1β, TGF-β1, and HIF-1α and increased TGF-β3. Therefore, the combination of therapeutic ultrasound and NMES is the most potent rehabilitation program for treating quadriceps contracture. [ABSTRACT FROM AUTHOR]

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutation in the dystrophin gene. Currently there is no cure for DMD. We introduced a novel human Dystrophin Expressing Chimeric (DEC) cell therapy of myoblast origin and confirmed the safety and efficacy of DEC in the mdx mouse models of DMD. In this study, we assessed histological and morphological changes in the cardiac, diaphragm, and gastrocnemius muscles of the mdx/scid mice after the transplantation of human DEC therapy via the systemic-intraosseous route. The efficacy of different DEC doses was evaluated at 90 days (0.5 × 106 and 1 × 106 DEC cells) and 180 days (1 × 106 and 5 × 106 DEC cells) after administration. The evaluation of Hematoxylin & Eosin (H&E)-stained sectional slices of cardiac, diaphragm, and gastrocnemius muscles included assessment of muscle fiber size by minimal Feret's diameter method using ImageJ software. The overall improvement in muscle morphology was observed in DMD-affected target muscles in both studies, as evidenced by a shift in fiber size distribution toward the wild type (WT) phenotype and by an increase in the mean Feret's diameter compared to the vehicle-injected controls. These findings confirm the long-term efficacy of human DEC therapy in the improvement of overall morphological pathology in the muscles affected by DMD and introduce DEC as a novel therapeutic approach for DMD patients. [ABSTRACT FROM AUTHOR]

Abstract Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age‐related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine‐Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age‐related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients.

Botulinum Neurotoxin Type A (BoNT-A) injections using Ultrasound (US) guidance have led to research evaluating changes in muscle architecture. Controversy remains as to what constitutes increased Echo-Intensity (EI) in spastic muscles and whether this may affect outcomes. We aim to provide a narrative review of US muscle architecture changes following Central Nervous System (CNS) lesions and explore their relationship to spasticity. Medline, CINAHL, and Embase databases were searched with keywords: ultrasonography, hypertonia, spasticity, fibrosis, and Heckmatt. Three physicians reviewed the results of the search to select relevant papers. Reviews identified in the search were used as a resource to identify additional studies. A total of 68 papers were included. Four themes were identified, including histopathological changes in spastic muscle, effects of BoNT-A on the muscle structure, available US modalities to assess the muscle, and utility of US assessment in clinical spasticity. Histopathological studies revealed atrophic and fibro-fatty changes after CNS lesions. Several papers described BoNT-A injections contributing to those modifications. These changes translated to increased EI. The exact significance of increased muscle EI remains unclear. The Modified Heckmatt Scale (MHS) is a validated tool for grading muscle EI in spasticity. The use of the US may be an important tool to assess muscle architecture changes in spasticity and improve spasticity management. Treatment algorithms may be developed based on the degree of EI. Further research is needed to determine the incidence and impact of these EI changes in spastic muscles.

Adipose-derived stem cells (ADSC) therapy shows promise as an effective treatment for dystrophinopathy. Fibro-/adipogenic progenitors (FAPs) play an essential role in the myogenesis of muscle satellite cells and contribute to muscle fibrosis and adipocyte infiltration. The interleukin 4 (IL-4) pathway acts as a switch that regulates the functions of FAPs. The interaction between FAPs and engrafted cells remains unclear. In this study, we used a co-culture system to investigate possible crosstalk between the FAPs of dystrophic mice and ADSC overexpressing IL4 (IL4-ADSC) and control ADSC. Systemic transplantation of IL4-ADSC and control ADSC in dystrophic mice was conducted for 16 weeks, after which motor function and molecular improvements were evaluated. Overexpression of IL4 in ADSC significantly promoted myogenesis in vitro, increasing the expression of Pax7, Myogenin, and MyHC. Co-culture indicated that although myoblasts derived from control ADSC promoted adipogenic and fibrogenic differentiation of FAPs, FAPs did not significantly affect myogenesis of ADSC-derived myoblasts. However, overexpression of IL4 in ADSC inhibited their myotube-dependent promotion of FAPs differentiation on the one hand and promoted FAPs to enhance myogenesis on the other. Dystrophic mice administered with IL4-ADSC-derived myoblasts displayed significantly better motor ability, more engrafted cells showing dystrophin expression, and less muscle fibrosis, intramuscular adipocytes, and macrophage infiltration than mice administered control-ADSC-derived myoblasts. In conclusion, IL4 activation enhanced the therapeutic potential of ADSC transplantation in dystrophic mice, possibly by improving the myogenesis of IL4-ADSC and altering the crosstalk between engrafted stem cells and resident FAPs. [ABSTRACT FROM AUTHOR]

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant epigenetic disorder with highly variable muscle involvement and disease progression. Ongoing clinical trials, aimed at counteracting muscle degeneration and disease progression in FSHD patients, increase the need for reliable biomarkers. Muscle magnetic resonance imaging (MRI) studies showed that the appearance of STIR-positive (STIR+) lesions in FSHD muscles represents an initial stage of muscle damage, preceding irreversible adipose changes. Our study aimed to investigate fibrosis, a parameter of muscle degeneration undetectable by MRI, in relation to disease activity and progression of FSHD muscles. We histologically evaluated collagen in FSHD1 patients’ (STIR+ n = 27, STIR− n = 28) and healthy volunteers’ (n = 12) muscles by picrosirius red staining. All patients (n = 55) performed muscle MRI before biopsy, 45 patients also after 1 year and 36 patients also after 2 years. Fat content (T1 signal) and oedema/inflammation (STIR signal) were evaluated at baseline and at 1- and 2-year MRI follow-up. STIR+ muscles showed significantly higher collagen compared to both STIR− (p = 0.001) and healthy muscles (p

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant epigenetic disorder with highly variable muscle involvement and disease progression. Ongoing clinical trials, aimed at counteracting muscle degeneration and disease progression in FSHD patients, increase the need for reliable biomarkers. Muscle magnetic resonance imaging (MRI) studies showed that the appearance of STIR-positive (STIR+) lesions in FSHD muscles represents an initial stage of muscle damage, preceding irreversible adipose changes. Our study aimed to investigate fibrosis, a parameter of muscle degeneration undetectable by MRI, in relation to disease activity and progression of FSHD muscles. We histologically evaluated collagen in FSHD1 patients’ (STIR+ n = 27, STIR− n = 28) and healthy volunteers’ (n = 12) muscles by picrosirius red staining. All patients (n = 55) performed muscle MRI before biopsy, 45 patients also after 1 year and 36 patients also after 2 years. Fat content (T1 signal) and oedema/inflammation (STIR signal) were evaluated at baseline and at 1- and 2-year MRI follow-up. STIR+ muscles showed significantly higher collagen compared to both STIR− (p = 0.001) and healthy muscles (p < 0.0001). STIR− muscles showed a higher collagen content compared to healthy muscles (p = 0.0194). FSHD muscles with a worsening in fatty infiltration during 1- (P = 0.007) and 2-year (P < 0.0001) MRI follow-up showed a collagen content of 3.6- and 3.7-fold higher compared to FSHD muscles with no sign of progression. Moreover, the fibrosis was significantly higher in STIR+ muscles who showed a worsening in fatty infiltration in a timeframe of 2 years compared to both STIR− (P = 0.0006) and STIR+ muscles with no sign of progression (P = 0.02). Fibrosis is a sign of muscle degeneration undetectable at MRI never deeply investigated in FSHD patients. Our data show that 23/27 of STIR+ and 12/28 STIR− muscles have a higher amount of collagen deposition compared to healthy muscles. Fibrosis is higher in FSHD muscles with a worsening in fatty infiltration thus suggesting that its evaluation with innovative non-invasive techniques could be a candidate prognostic biomarker for FSHD, to be used to stratify patients and to evaluate the efficacy of therapeutic treatments. [ABSTRACT FROM AUTHOR]

The matricellular protein cell communication factor 2/connective tissue growth factor (CCN2/CTGF) is critical to development of neuromuscular fibrosis. Here, we tested whether anti-CCN2 antibody treatment will reduce established forepaw fibro-degenerative changes and improve function in a rat model of overuse injury. Adult female rats performed a high repetition high force (HRHF) task for 18 weeks. Tissues were collected from one subset after 18 wks (HRHF-Untreated). Two subsets were provided 6 wks of rest with concurrent treatment with anti-CCN2 (HRHF-Rest/anti-CCN2) or IgG (HRHF-Rest/IgG). Results were compared to IgG-treated Controls. Forepaw muscle fibrosis, neural fibrosis and entheseal damage were increased in HRHF-Untreated rats, compared to Controls, and changes were ameliorated in HRHF-Rest/anti-CCN2 rats. Anti-CCN2 treatment also reduced phosphorylated-β-catenin (pro-fibrotic protein) in muscles and distal bone/entheses complex, and increased CCN3 (anti-fibrotic) in the same tissues, compared to HRHF-Untreated rats. Grip strength declines and mechanical sensitivity observed in HRHF-Untreated improved with rest; grip strength improved further in HRHF-Rest/anti-CCN2. Grip strength declines correlated with muscle fibrosis, entheseal damage, extraneural fibrosis, and decreased nerve conduction velocity, while enhanced mechanical sensitivity (a pain-related behavior) correlated with extraneural fibrosis. These studies demonstrate that blocking CCN2 signaling reduces established forepaw neuromuscular fibrosis and entheseal damage, which improves forepaw function, following overuse injury. [ABSTRACT FROM AUTHOR]

Quadriceps contracture is a condition where the muscle–tendon unit is abnormally shortened. The treatment prognosis is guarded to poor depending on the progress of the disease. To improve the prognosis, we investigated the effectiveness of therapeutic ultrasound and NMES in treating quadriceps contracture in an immobilized rat model. Thirty-six Wistar rats were randomized into control, immobilization alone, immobilization and spontaneous recovery, immobilization and therapeutic ultrasound, immobilization and NMES, and immobilization and therapeutic ultrasound and NMES combination groups. The continuous therapeutic ultrasound (frequency, 3 MHz, intensity 1 W/cm2) and NMES (TENS mode, frequency 50 Hz; intensity 5.0 ± 0.8 mA) were performed on the quadriceps muscle. On Day 15, immobilization-induced quadriceps contracture resulted in a decreased ROM of the stifle joint, reduction in the sarcomere length, muscle atrophy, and muscle fibrosis. On Day 43, therapeutic ultrasound, NMES, and combining both methods improved muscle atrophy and shortening and decreased collagen type I and III and α-SMA protein. The combination of therapeutic ultrasound and NMES significantly reduced the mRNA expression of IL-1β, TGF-β1, and HIF-1α and increased TGF-β3. Therefore, the combination of therapeutic ultrasound and NMES is the most potent rehabilitation program for treating quadriceps contracture.

Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and ultrastructural changes of muscle fibers in sarcopenia, mitochondria and their quality control pathways play relevant roles. Mononucleated muscle stem cells/satellite cells (MSCs) have been attributed a critical role in muscle repair after an injury. The involvement of mitochondria in supporting MSC-directed muscle repair is unclear. There is evidence that a reduction in mitochondrial biogenesis blunts muscle repair, thus indicating that the delivery of functional mitochondria to injured muscles can be harnessed to limit muscle fibrosis and enhance restoration of muscle function. Injection of autologous respiration-competent mitochondria from uninjured sites to damaged tissue has been shown to reduce infarct size and enhance cell survival in preclinical models of ischemia–reperfusion. Furthermore, the incorporation of donor mitochondria into MSCs enhances lung and cardiac tissue repair. This strategy has also been tested for regeneration purposes in traumatic muscle injuries. Indeed, the systemic delivery of mitochondria promotes muscle regeneration and restores muscle mass and function while reducing fibrosis during recovery after an injury. In this review, we discuss the contribution of altered MSC function to sarcopenia and illustrate the prospect of harnessing mitochondrial delivery and restoration of MSCs as a therapeutic strategy against age-related sarcopenia.

The matricellular protein cell communication factor 2/connective tissue growth factor (CCN2/CTGF) is critical to development of neuromuscular fibrosis. Here, we tested whether anti-CCN2 antibody treatment will reduce established forepaw fibro-degenerative changes and improve function in a rat model of overuse injury. Adult female rats performed a high repetition high force (HRHF) task for 18 weeks. Tissues were collected from one subset after 18 wks (HRHF-Untreated). Two subsets were provided 6 wks of rest with concurrent treatment with anti-CCN2 (HRHF-Rest/anti-CCN2) or IgG (HRHF-Rest/IgG). Results were compared to IgG-treated Controls. Forepaw muscle fibrosis, neural fibrosis and entheseal damage were increased in HRHF-Untreated rats, compared to Controls, and changes were ameliorated in HRHF-Rest/anti-CCN2 rats. Anti-CCN2 treatment also reduced phosphorylated-β-catenin (pro-fibrotic protein) in muscles and distal bone/entheses complex, and increased CCN3 (anti-fibrotic) in the same tissues, compared to HRHF-Untreated rats. Grip strength declines and mechanical sensitivity observed in HRHF-Untreated improved with rest; grip strength improved further in HRHF-Rest/anti-CCN2. Grip strength declines correlated with muscle fibrosis, entheseal damage, extraneural fibrosis, and decreased nerve conduction velocity, while enhanced mechanical sensitivity (a pain-related behavior) correlated with extraneural fibrosis. These studies demonstrate that blocking CCN2 signaling reduces established forepaw neuromuscular fibrosis and entheseal damage, which improves forepaw function, following overuse injury.

The collagenous extracellular matrix (ECM) of skeletal muscle functions to transmit force, protect sensitive structures, and generate passive tension to resist stretch. The mechanical properties of the ECM change with age, atrophy, and neuromuscular pathologies, resulting in an increase in the relative amount of collagen and an increase in stiffness. Although numerous studies have focused on the effect of muscle fibrosis on passive muscle stiffness, few have examined how these structural changes may compromise contractile performance. Here we combine a mathematical model and experimental manipulations to examine how changes in the mechanical properties of the ECM constrain the ability of muscle fibers and fascicles to radially expand and how such a constraint may limit active muscle shortening. We model the mechanical interaction between a contracting muscle and the ECM using a constant volume, pressurized, fiber-wound cylinder. Our model shows that as the proportion of a muscle cross section made up of ECM increases, the muscle's ability to expand radially is compromised, which in turn restricts muscle shortening. In our experiments, we use a physical constraint placed around the muscle to restrict radial expansion during a contraction. Our experimental results are consistent with model predictions and show that muscles restricted from radial expansion undergo less shortening and generate less mechanical work under identical loads and stimulation conditions. This work highlights the intimate mechanical interaction between contractile and connective tissue structures within skeletal muscle and shows how a deviation from a healthy, well-tuned relationship can compromise performance.

Non-Progressive Congenital Myopathy is a disease characterized by muscle weakness, and unfortunately, there is no conventional treatment. In the last decade, regenerative medicine practices have become a rising value, and Mesenchymal Stem Cells (MSCs) have fascinating outcomes in regenerative medicine with their high regenerative capacities, their ability to regulate with paracrine secretions, and their immunological properties. Based on our experience in our previous clinical studies, Wharton's-Jelly-derived (WJ-)MSCs are the most suitable source for muscle diseases among all MSC sources. In this study, we evaluated the outcomes of 10 doses of WJ-MSC transplantation to the patient diagnosed with Non-Progressive Congenital Myopathy. A 17-year-old female with a SPEN-1 mutation, Non-Progressive Congenital Myopathy patient received 10 times as 1×106/kg in the intra-arterial, intramuscular and intravenous administration of allogenic WJ-MSC. Before and after the treatment, the patient was followed-up with the upper extremity scale, Vignos lower extremity scale, muscle strength scale, functional independence measure, and evaluation of Serum creatine kinase (CK) levels. Improvement in both upper extremity scale and Vignos lower extremity scales, increasing in muscle strength, and decreasing in CK-level were detected. Although transplantation of WJ-MSC cannot treat any genetic-based diseases, they may benefit in alleviating clinical outcomes of disease. More importantly, WJ-MSC transplantation may offer a better quality of life by alleviating the symptoms of this rare disease with no treatment option that can be provided in conventional methods. [ABSTRACT FROM AUTHOR]

Non-Progressive Congenital Myopathy is a disease characterized by muscle weakness, and unfortunately, there is no conventional treatment. In the last decade, regenerative medicine practices have become a rising value, and Mesenchymal Stem Cells (MSCs) have fascinating outcomes in regenerative medicine with their high regenerative capacities, their ability to regulate with paracrine secretions, and their immunological properties. Based on our experience in our previous clinical studies, Wharton's-Jelly-derived (WJ-)MSCs are the most suitable source for muscle diseases among all MSC sources. In this study, we evaluated the outcomes of 10 doses of WJ-MSC transplantation to the patient diagnosed with Non-Progressive Congenital Myopathy. A 17-year-old female with a SPEN-1 mutation, Non-Progressive Congenital Myopathy patient received 10 times as 1×10⁶/kg in the intra-arterial, intramuscular and intravenous administration of allogenic WJ-MSC. Before and after the treatment, the patient was followed-up with the upper extremity scale, Vignos lower extremity scale, muscle strength scale, functional independence measure, and evaluation of Serum creatine kinase (CK) levels. Improvement in both upper extremity scale and Vignos lower extremity scales, increasing in muscle strength, and decreasing in CK-level were detected. Although transplantation of WJ-MSC cannot treat any genetic-based diseases, they may benefit in alleviating clinical outcomes of disease. More importantly, WJ-MSC transplantation may offer a better quality of life by alleviating the symptoms of this rare disease with no treatment option that can be provided in conventional methods.

Dysregulation and fibrosis of the extracellular matrix (ECM) in skeletal muscle is a consequence of injury. Current ECM assessment necessitates muscle biopsies to evaluate alterations to the muscle ECM, which is often not practical in humans. The goal of this study was to evaluate the potential of a magnetic resonance imaging sequence that quantifies T1ρ relaxation time to predict ECM collagen composition and organization. T1ρ imaging was performed and muscle biopsies obtained from the involved and non‐involved vastus lateralis muscle on 27 subjects who had an anterior cruciate ligament (ACL) tear. T1ρ times were quantified via monoexponential decay curve fitted to a series of T1ρ‐weighted images. Several ECM indices, including collagen content and organization, were obtained using immunohistochemistry and histochemistry in addition to hydroxyproline. Model selection with multiple linear regression was used to evaluate the relationships between T1ρ times and ECM composition. Additionally, the ACL‐deficient and healthy limb were compared to determine sensitivity of T1ρ to detect early adaptations in the muscle ECM following injury. We show that T1ρ relaxation time was strongly associated with collagen unfolding (t = 4.093, P = 0.0007) in the ACL‐deficient limb, and collagen 1 abundance in the healthy limb (t = 2.75, P = 0.014). In addition, we show that T1ρ relaxation time is significantly longer in the injured limb, coinciding with significant differences in several indices of collagen content and remodelling in the ACL‐deficient limb. These results support the use of T1ρ to evaluate ECM composition in skeletal muscle in a non‐invasive manner. Key points: Dysregulation and fibrotic transformation of the skeletal muscle extracellular matrix (ECM) is a common pathology associated with injury and ageing.Studies of the muscle ECM in humans have necessitated the use of biopsies, which are impractical in many settings.Non‐invasive MRI T1ρ relaxation time was validated to predict ECM collagen composition and organization with aligned T1ρ imaging and biopsies of the vastus lateralis in the healthy limb and anterior cruciate ligament (ACL)‐deficient limb of 27 subjects.T1ρ relaxation time was strongly associated with collagen abundance and unfolding in the ACL‐deficient limb, and T1ρ relaxation time was strongly associated with total collagen abundance in the healthy limb.T1ρ relaxation time was significantly longer in the ACL‐deficient limb, coinciding with significant increases in several indices of muscle collagen content and remodelling supporting the use of T1ρ to non‐invasively evaluate ECM composition and pathology in skeletal muscle. [ABSTRACT FROM AUTHOR]

Rotator cuff tears can cause irreversible changes (e.g., fibrosis) to the structure and function of the injured muscle(s). Fibrosis leads to increased muscle stiffness resulting in increased tension at the rotator cuff repair site. This tension influences repairability and healing potential in the clinical setting. However, the micro- and meso-scale structural and molecular sources of these whole-muscle mechanical changes are poorly understood. Here, single muscle fiber and fiber bundle passive mechanical testing was performed on rat supraspinatus and infraspinatus muscles with experimentally induced massive rotator cuff tears (Tenotomy) as well as massive tears with chemical denervation (Tenotomy + BTX) at 8 and 16 weeks post-injury. Titin molecular weight, collagen content, and myosin heavy chain profiles were measured and correlated with mechanical variables. Single fiber stiffness was not different between controls and experimental groups. However, fiber bundle stiffness was significantly increased at 8 weeks in the Tenotomy + BTX group compared to Tenotomy or control groups. Many of the changes were resolved by 16 weeks. Only fiber bundle passive mechanics was weakly correlated with collagen content. These data suggest that tendon injury with concomitant neuromuscular compromise results in extra-cellular matrix production and increases in stiffness of the muscle, potentially complicating subsequent attempts for surgical repair.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL‐10 and TNFα were significantly elevated post injury, indicating toll‐like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL‐10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA‐approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short‐term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury. [ABSTRACT FROM AUTHOR]

Abstract Background Lack of animal model of radiation induced muscle fibrosis, this study aimed to establish such a model by using 90 Gy single dose irradiation to mimic clinical relevance and also to explore the potential post-irradiation regenerative mechanism. Methods SD rats were randomly divided into dose investigation groups and time gradient groups. Group1–6 were irradiated with a single dose of 65Gy, 70Gy, 75Gy, 80Gy, 85Gy and 90Gy respectively, and the degree of rectus femoris fibrosis in the irradiated area was detected at 4 weeks after irradiation. Group 7–9 were irradiated with a single dose of 90Gy, and the results were detected 1, 2, 4, and 8 weeks after irradiation. Then the general condition of rats was recorded. Masson staining was used to detect muscle fibrosis. The ultrastructure of muscles was observed by electron microscope, and the expression changes of satellite cell proliferation and differentiation related genes were detected by quantitative real-time-PCR. Results A single dose of 90Gy irradiation could cause muscle fibrosis in rats. As time goes on, the severity of muscle fibrosis and the expression of TGF- β1 increased. Significant swelling of mitochondria, myofilament disarrangement and dissolution, obvious endothelial cell swelling, increased vascular permeability, decrease of blood cell, deposition of fibrosis tissue around the vessel could be found compared with the control group. At around the 4th week, the expressions of Pax7, Myf5, MyoD, MyoG, Mrf4 increased. Conclusion Irradiation of 90Gy can successfully establish the rat model of radiation-induced muscle fibrosis. This model demonstrated that regenerative process was initiated by the irradiation only at an early stage, which can serve a suitable model for investigating regenerative therapy for post-radiation muscle fibrosis.

Severe burn injury induces a myriad of deleterious effects to skeletal muscle, resulting in impaired function and delayed recovery. Following burn, catabolic signaling and myofiber atrophy are key fiberintrinsic determinants of weakness; less well understood are alterations in the interstitial environment surrounding myofibers. Muscle quality, specifically alterations in the extracellular matrix (ECM), modulates force transmission and strength. We sought to determine the impact of severe thermal injury on adaptation to the muscle ECM and quantify muscle fibrotic burden. After a 30% total body surface area dorsal burn, spinotrapezius muscle was harvested from mice at 7 (7d, n = 5), 14 (14d, n = 4), and 21 days (21d, n = 4), and a sham control group was also examined (Sham, n = 4). Expression of transforming growth factor-β (TGFβ), myostatin, and downstream effectors and proteases involved in fibrosis and collagen remodeling were measured by immunoblotting, and immunohistochemical and biochemical analyses assessed fibrogenic cell abundance and collagen deposition. Myostatin signaling increased progressively through 21 days postburn alongside fibrogenic/adipogenic progenitor cell expansion, with abundance peaking at 14 days postburn. Postburn, elevated expression of tissue inhibitor of matrix metalloproteinase 1 supported collagen remodeling resulting in a net accumulation of muscle collagen content. Collagen accumulation peaked at 14 days postburn but remained elevated through 21 days postburn, demonstrating minimal resolution of burn-induced fibrosis. These findings highlight a progressive upregulation of fibrogenic processes following burn injury, eliciting a fibrotic muscle phenotype that hinders regenerative capacity and is not resolved with 21 days of recovery. [ABSTRACT FROM AUTHOR]

Hypoxia refers to the decrease in oxygen tension in the tissues, and the central effector of the hypoxic response is the transcription factor Hypoxia-Inducible Factor α (HIF1-α). Transient hypoxia in acute events, such as exercising or regeneration after damage, play an important role in skeletal muscle physiology and homeostasis. However, sustained activation of hypoxic signaling is a feature of skeletal muscle injury and disease, which can be a consequence of chronic damage but can also increase the severity of the pathology and worsen its outcome. Here, we review evidence that supports the idea that hypoxia and HIF-1α can contribute to the establishment of fibrosis in skeletal muscle through its crosstalk with other profibrotic factors, such as Transforming growth factor β (TGF-β), the induction of profibrotic cytokines expression, as is the case of Connective Tissue Growth Factor (CTGF/CCN2), or being the target of the Renin-angiotensin system (RAS). [ABSTRACT FROM AUTHOR]

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies. [ABSTRACT FROM AUTHOR]

We investigated the effects of cyclic muscle twitch contraction caused by neuromuscular electrical stimulation (NMES) on immobilization-induced muscle contracture and fibrosis in rats. Twenty-nine rats were divided into control, immobilization, and immobilization with muscle contraction groups. The ankle joints of the immobilization and muscle contraction rats were fixed in full plantar flexion with a plaster cast for 4 weeks. In the muscle contraction group, cyclic muscle twitch contraction of the soleus muscle was induced using a commercial device (1 Hz, 4 ± 2 mA, 60 min/day, 5 times/week) with the ankle joint immobilized. The dorsiflexion range of ankle joint motion in the muscle contraction group was significantly greater than that in the immobilization group. The expressions of fibrosis-related genes (i.e., hypoxia inducible factor-1α, transforming growth factor-β1, α-smooth muscle actin, and types I and III collagen) were significantly decreased in the muscle contraction group compared to the immobilization group. The fluorescence intensities of type I and type III collagen in the perimysium and endomysium in the muscle contraction group were significantly decreased compared to the immobilization group. These results suggest that cyclic muscle twitch contraction induced by NMES might alleviate skeletal muscle fibrosis, reducing immobilization-induced muscle contracture. [ABSTRACT FROM AUTHOR]

Volumetric muscle loss (VML) injuries present a complex and heterogeneous clinical problem that results in a chronic loss of muscle tissue and strength. The primary limitation to muscle tissue regeneration after VML injury is the frank loss of all native muscle constituents in the defect, especially satellite cells and the basal lamina. Recent advancements in regenerative medicine have set forth encouraging and emerging translational and therapeutic options for these devastating injuries including the surgical implantation of acellular biological scaffolds. While these biomaterials can modulate the wound environment, the existing data do not support their capacity to promote appreciable muscle fiber regeneration that can contribute to skeletal muscle tissue functional improvements. An apparent restriction of endogenous satellite cell (i.e., pax7 + ) migration to acellular biological scaffolds likely underlies this deficiency. This work critically evaluates the role of an acellular biological scaffold in orchestrating skeletal muscle tissue regeneration, specifically when used as a regenerative medicine approach for VML injury. [ABSTRACT FROM AUTHOR]

The Sgcb-null mouse, with knocked-down β-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-β1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-β1 was present in areas of degeneration-regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears more promising than the mdx mouse for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments. Highlights • The Sgcb-null mouse develops severe muscular dystrophy, the mdx mouse does not. • Fibrosis developed earlier in Sgcb-null quadriceps and diaphragm than mdx. • Macrophages were commoner in mildly inflamed parts of Sgcb-null quadriceps than mdx. • The Sgcb-null model appears more useful than mdx for studying fibrotic mechanisms. • The Sgcb-null model also appears more useful for developing anti-fibrotic treatments. [ABSTRACT FROM AUTHOR]