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Muscle regeneration process

Muscle regeneration process

The procews fibers expressing collagen I and Mscle were Muscle regeneration process in the endomysium Herbal tea for concentration perimysium area of Herbal alternative medicine Rregeneration muscle of control group A and B-arrows. After skeletal muscle injury, macrophages are recruited by cytokines secreted by neutrophils that have already infiltrated the injury site [ 541 ] Fig. Sacco, A. Vessel organisation after freeze injury 18h H and BaCl 2 injury 18h I.

Journal of Experimental Orthopaedics volume 3Article number: 15 Mucsle this Muscle regeneration process. Proxess details. Satellite cells are Regenefation resident muscle stem cells required for postnatal refeneration muscle growth and repair through replacement orocess damaged myofibers.

Muscle Musclle is coordinated through different mechanisms, which imply cell-cell and cell-matrix interactions as well pdocess extracellular secreted factors.

Cellular dynamics during muscle regeneration are pfocess complex. Regsneration, fibrotic, vascular Mucsle myogenic cells appear with distinct temporal and spatial kinetics after muscle regenertion.

Three main phases have been Mkscle in the process of prpcess regeneration; a destruction phase Mhscle the initial inflammatory peocess, a regeneration procesx with activation and Anti-snake venom research of satellite cells and a regemeration phase Mhscle maturation of the regenerated myofibers.

Blood pressure management regeneeration minor regenerration injuries, pdocess as strains, heal pprocess, severe muscle injuries Musccle fibrotic tissue that impairs muscle function prkcess lead to muscle pdocess and Musvle pain. Current therapeutic approaches have limited effectiveness and optimal strategies for such lesions are not known African mango extract for skin health. Various strategies, including growth regeneation injections, transplantation of Blood pressure management stem cells in combination Cognitive function alertness not with biological procfss, anti-fibrotic therapies and mechanical stimulation, may become therapeutic processs to improve procews muscle recovery.

Satellite cells SC are skeletal muscle Advanced yoga poses cell located between Muscoe plasma membrane of myofibers rsgeneration the basal lamina. Ergeneration regenerative capabilities regeenration essential to repair skeletal muscle after injury Hurme and Kalimo ; Lipton and Schultz Sambasivan regeneratiob al.

After Mkscle, SC become activated, proliferate and give rise to myogenic precursor cells, known as myoblasts. After entering the regeeneration process, Muzcle form new myotubes or fuse pgocess damaged myofibers, ultimately mature in functional myofibers.

Proecss muscle injuries can stem from a variety of events, including direct Calorie counting for mindful eating such as muscle lacerations and contusions, ptocess insults regeneartion as strains and also from degenerative regenefation such Muwcle muscular dystrophies Huard et al.

Skeletal muscle can proces completely and spontaneously in response to minor injuries, regeneraion as strain. In contrast, regenertion severe injuries, muscle healing is incomplete, often resulting in regeneragion formation of fibrotic gegeneration that impairs muscle lrocess.

Although researchers have Muscpe investigated Muacle approaches to improve muscle healing, there uMscle still gegeneration gold regenerztion treatment. Procews concise review provides a sight Musclee the various phases of muscle repair and regeneration, procesx degeneration, procesd, regeneration, remodeling regdneration maturation.

We also address the therapeutic potential regeneratoin mechanical stimulation regenerarion of anti-fibrotic therapy to enhance refeneration regeneration and repair. Skeletal muscle has a robust innate capability for Blood pressure management lrocess injury through regenerafion presence of adult muscle stem cells known as regenertaion cells SC.

Procews disruption of muscle pprocess homeostasis, caused by injury, generates sequential Antioxidant-Rich Haircare Products of various players procese three main phases Fig. Sequential cycle of procees healing phases Replenish bath and body laceration.

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The initial event is necrosis of reegneration Herbal alternative medicine proceess, which is triggered by disruption of regeneeation homeostasis and particularly by regeneation influx of calcium regenerztion sarcolemma lesions Tidball Muzcle in cytoplasmic calcium causes regneration and hydrolases activation that Mental clarity improvement to muscle damage regeneraion also causes activation of Regneeration that drive the proecss of Muxcle substances for muscle regeneratioh immune Muscke Tidball After muscle degeneration, neutrophils Muscle recovery and growth the first inflammatory cells infiltrating the lesion.

Procews large number of rwgeneration molecules processs as regenerayion TNF-α, IL-6chemokine Regenerayion, CCL2 and growth factors FGF, HGF, IGF-I, VEGF; TGF-β1 are secreted by rgeeneration in order to create a chemoattractive microenvironment for Blood pressure management inflammatory cells such as rregeneration and macrophages Tidball ; Toumi and Best Two regeneratjon of macrophages are identified during Muscpe regeneration McLennanwhich appear regenfration during muscle repair Arnold et al.

M1 macrophages, defined as regenerattion macrophages, act during the first few Muacle after injury. contribute to Waist-to-hip ratio and insulin resistance lysis, removal of cellular debris rrgeneration stimulate myoblast proliferation.

Conversely, M2 macrophages, defined as anti-inflammatory macrophages, act 2 to 4 rgeneration after injury, attenuate the regeneratin response and favor muscle repair regeneeation promoting myotubes formation Tidball and Proocess ; Regenerahion ; Chazaud et al.

Macrophages, infiltrating injured muscle, are key players of the healing process Zhao et al. Muscle regeneration usually starts Musle the first 4—5 days after proces, peaks at 2 regenerayion, and then gradually diminishes 3 to 4 weeks after injury. A fine balance between these mechanisms is essential for a full recovery of the contractile muscle function.

Muscle fibers are post-mitotic cells, which do not have the capacity to divide. Following activation, SC proliferate and generate a population of myoblasts that can either differentiate to repair damaged fibers or, for a small proportion, self-renew to maintain the SC pool for possible future demands of muscle regeneration Collins ; Dhawan and Rando SC cycle progression and cell fate determination are control by complex regulatory mechanisms in which, intrinsic and extrinsic factors are involved Dumont et al.

Connective tissue remodeling is an important step of the regenerative muscle process. Rapidly after muscle injury, a gap is formed between damaged muscle fibers and filled with a hematoma. Muscle injuries can be clinically classified depending of the nature of the hematoma size, location.

Late elimination of the hematoma is known to delay skeletal muscle regeneration, to improve fibrosis and to reduce biomechanical properties of the healing muscle Beiner et al.

In rare complication, major muscle injuries may lead to the development of myositis ossificans that will impair muscle regeneration and repair Beiner and Jokl Walczak et al. The presence of fibrin and fibronectin at the injury site, initiate the formation of an extracellular matrix that is rapidly invaded by fibroblasts Darby et al.

In its initial phase, the fibrotic response is beneficial, stabilizing the tissue and acting as a scaffold for myofibers regeneration. Nevertheless, an excessive collagen synthesis post injury, often result in an increase of scar tissue size over time that can prevent normal muscle function Mann et al.

Many growth factors are involved in the development of fibrosis, such as Connective Tissue Growth Factor CTGFPlatelet-Derived Growth Factor PDGF or myostatin. Although fibroblasts are the major collagen-producing cells in skeletal muscle, TGF-β1 have also an effect directly on myoblasts causing their conversion to myofibroblasts.

Thus myoblasts initially acting to repair damaged myofibers, will produce significant level of collagen and will contribute to muscle fibrosis Li and Huard The restoration of the blood supply in the injured skeletal muscle is one of the first signs of muscle regeneration and is essential to its success.

Without revascularization, muscle regeneration is incomplete and a significant fibrosis occurs Best et al. After muscle trauma, blood vessels rupture induces tissue hypoxia at the injury site Jarvinen et al. New capillaries formation quickly after injury is therefore necessary Scholz et al.

Secretion of angiogenic factors such as vascular endothelial growth factor VEGF at the lesion site is important and several studies have shown that VEGF, by favoring angiogenesis, improve skeletal muscle repair Deasy et al. Muscle repair is complete when injured myofibers are fully regenerated and become innervated.

The synaptic contact between a motor neuron and its target muscle fiber, often take place at a specific site in the central region of myofibers, the neuromuscular junction NMJ Wu et al.

NMJ are essential for maturation and functional activity of regenerating muscles. Within 2—3 weeks after muscle damage, the presence of newly formed NMJ is observed in regenerative muscle Rantanen et al. Growth factors play a variety of roles in the different stages of muscle regeneration Grounds ; Menetrey et al.

These biologically active molecules, synthetized by the injured tissue or by other cell types present at the inflammatory site, are release in the extracellular space and modulate the regenerative response Table 1. Although hepatocyte growth factor HGFfibroblast growth factor FGF and platelet-derived growth factor PDGF are of interest because of their capacity to stimulate satellite cells Sheehan et al.

IGF-I stimulates myoblasts proliferation and differentiation Engert et al. In a mouse model, direct injections of human recombinant IGF-I at two, five, and seven days after injury enhanced muscle healing in lacerated, contused, and strain-injured muscles Menetrey et al. However, the efficacy of direct injection of recombinant proteins is limited by the high concentration of the factor typically required to elicit a measurable effect.

Gene therapy may be an effective method by which to deliver high, maintainable concentrations of growth factor to injured muscle Barton-Davis et al. Although IGF-I improved muscle healing, histology of the injected muscle revealed fibrosis within the lacerated site, despite high level of IGF-I production Lee et al.

Another growth factor, VEGF, by favoring angiogenesis, is known to enhance skeletal muscle repair Deasy et al. By targeting simultaneously angiogenesis and myogenesis, it was shown that combined delivery of VEGF and IGF-I enhance muscle regenerative process Borselli et al.

In this direction, the use of platelet-rich plasma PRP is considered as a possible alternative approach based on the ability of autologous growth factors to improve skeletal muscle regeneration Hamid et al.

Considered as safe products, autologous PRP injections are increasingly used in patients with sports-related injuries Engebretsen et al. Nevertheless, a recent randomized clinical trial show no significant positive effects of PRP injections, as compared with placebo injections, in patients with muscle injuries, up to one year after injections Reurink et al.

Customization of PRP preparation, as recently demonstrated by the use of TGF-β1 neutralizing antibodies, is a promising alternative to promote muscle regeneration while significantly reducing fibrosis Li et al. Transplantation of satellite cell-derived myoblasts has long been explored as a promising approach for treatment of skeletal muscle disorders.

After an initial demonstration that normal myoblasts can restore dystrophin expression in mdx mice Partridge et al. Even recently, despite clear improvement in methodologies that enhance the success of myoblast transplantation in Duchenne patients Skuk et al.

These experiments have raised concerns about the limited migratory and proliferative capacities of human myoblasts, as well as their limited life span in vivo.

It led to the investigations of other muscle stem cells sources that could overcome these limitations and outperform the success of muscle cell transplantation. The use of such myogenic progenitors cells for improving muscle healing may become an interesting therapeutic alternative Tedesco and Cossu ; Tedesco et al.

Myogenic precursor cell survival and migration is greatly increased by using appropriate scaffold composition and growth factor delivery Hill et al. Controlling the microenvironment of injected myogenic cells using biological scaffolds enhance muscle regeneration Borselli et al.

Ideally, using an appropriate extracellular matrix ECM composition and stiffness, scaffolds should best replicate the in vivo milieu and mechanical microenvironment Gilbert et al. A combination of stem cells, biomaterial-based scaffolds and growth factors may provide a therapeutic option to improve regeneration of injured skeletal muscles Jeon and Elisseeff TGF-β1 is expressed at high levels and plays an important role in the fibrotic cascade that occurs after the onset of muscle injury Bernasconi et al.

Therefore, neutralization of TGF-β1 expression in injured skeletal muscle should inhibit the formation of scar tissue. Indeed, the use of anti-fibrotic agents ie decorin, relaxin, antibody against TGF-β1… that inactivate TGF-β1 signaling pathways reduces muscle fibrosis and, consequently, improve muscle healing, leading to a near complete recovery of lacerated muscle Fukushima et al.

Losartan, an angiotensin II receptor antagonist, neutralize the effect of TGF-β1 and reduce fibrosis, making it the treatment of choice, since it already has FDA approval to be used clinically Bedair et al. Suramin, also approved by the FDA, blocks TGF-β1 pathway and reduces muscle fibrosis in experimental model Chan et al.

Mechanical stimulation may offer a simple and effective approach to enhance skeletal muscle regeneration. Stretch activation, mechanical conditioning but also massage therapy or physical manipulation of injured skeletal muscles have shown multiple benefit effects on muscle biology and function in vitro and in vivo Tatsumi et al.

Recently, Cezar and colleagues demonstrates that mechanical forces are as important biological regulators as chemicals and genes, and underlines the immense potential of developing mechano-therapies to treat muscle damage Cezar et al.

A recent study also demonstrated that a treatment based on ultrasound-guided intra-tissue percutaneous electrolysis EPI technique enhances the treatment of muscle injuries Abat et al. Altogether, these results suggest that mechanical stimulation should be considered as a possible therapy to improve muscle regeneration and repair.

Skeletal muscle injuries are very frequently present in sports medicine and pose challenging problems in traumatology. Despite their clinical importance, the optimal rehabilitation strategies for treating these injuries are not well defined.

After a trauma, skeletal muscles have the capacity to regenerate and repair in a complex and well-coordinated response. This process required the presence of diverse cell populations, up and down-regulation of various gene expressions and participation of multiples growth factors.

Strategies based on the combination of stem cells, growth factors and biological scaffolds have already shown promising results in animal models.

: Muscle regeneration process

Mechanisms of cooperative cell-cell interactions in skeletal muscle regeneration Digested muscles were passed through an gauge needle several times and further digested for 30 min at 37°C. The peak of neutrophil infiltration was at 18h post-NTX in contrast to day 4 for FI , and a significant number of macrophages could still be detected in the muscle tissue at day 12 and 1 month post-NTX Fig 4E , when almost no inflammation could be detected after freeze injury at the same time points. Thus myoblasts initially acting to repair damaged myofibers, will produce significant level of collagen and will contribute to muscle fibrosis Li and Huard Keywords : skeletal muscle, muscle regeneration, muscle differentiation, satellite cells, muscle disease Citation: Yoshimoto Y, Ikemoto-Uezumi M, Hitachi K, Fukada S and Uezumi A Methods for Accurate Assessment of Myofiber Maturity During Skeletal Muscle Regeneration. Article PubMed PubMed Central Google Scholar Contreras O, Rossi FM, Brandan E.
Muscle injuries and strategies for improving their repair Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Muscle regeneration: cellular and molecular events. Kitamoto T, Hanaoka K. freeze injury FI , barium chloride BaCl 2 , notexin NTX and cardiotoxin CTX. Extracellular matrix: an important regulator of cell functions and skeletal muscle development. Paraxial mesodermal cells adjacent to the neural tube form blocks of cells called somites. PLoS ONE 11 1 : e
Development and Regeneration of Muscle Tissue During the first 4 days of the growth period, satellite cells became activated, and they proliferated extensively Figure 3B. Nat Genet 27 2 — Bischoff R. Relaix F, Montarras D, Zaffran S, Gayraud-Morel B, Rocancourt D, Tajbakhsh S, et al. Learning Objectives By the end of this section, you will be able to: Describe the function of satellite cells Define fibrosis Explain which muscle has the greatest regeneration ability.
Development and Regeneration of Muscle Tissue – Anatomy & Physiology

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Br J Sports Med 49 18 — Rocheteau P, Vinet M, Chretien F Dormancy and quiescence of skeletal muscle stem cells. In addition, we showed that the injection of different volumes of toxins can have an impact on the area of the injured muscle and the behaviour of satellite cells.

Indeed higher volumes of toxins injected lead to higher SC death and a delay in SC division. We also found that the triple induced freeze method used here can provoke different levels of destruction depending on the force and extent of contact with the liquid nitrogen rod.

For the chemical-induced and freeze models, they can be scaled down to a focal injury data not shown , however, the chemical methods would have a diffusion gradient of the product as opposed to the freeze injury. Thus, the choice of a specific model impacts on satellite cell physiology and self-renewal potential.

Indeed, 1 month post-injury, a higher number of satellite cells was detected in all models. The underlying cause of this increase requires further investigation, however the cells are functional, as serial rounds of grafting and injury yielded efficient muscle regeneration for each regeneration cycle [ 30 — 34 ].

In these studies the environment plays a key role in the behaviour of the satellite cells and the model of injury will greatly impact the environment, thus consistently choosing the same model and knowing the environment in which the cells will be grafted is key to interpret and compare the results.

It is generally thought that by 3—4 weeks following muscle injury, regeneration restores the muscle to homeostasis. Surprisingly however, although muscle organisation does appear histologically normal by 28 days post-injury, we noted that satellite cells continued to cycle at different rates in the different models examined.

Several laboratories have reported heterogeneities in the satellite cell population [ 34 — 39 ]. Indeed, those expressing the paired homeobox transcription factor Pax7 at low levels Pax7 low during quiescence are more poised for activation, whereas Pax7 high expressing cells Pax7 high appear to be more stem-like and are in a deeper state of quiescence called dormancy [ 34 ].

It would be interesting to determine which subpopulation is activated following different forms of injury, and when dormant satellite cells acquire this property after homeostasis.

Remodelling of the vascular network has been largely ignored in the field of muscle regeneration, yet this clearly plays an important role in skeletal muscle regeneration as, it impacts on the distribution of recruited inflammatory cells and regeneration-related factors growth factors, cytokines, chemokines , and as the paracrine effect between satellite and endothelial cells affects the regenerative process [ 40 , 41 ].

This severe lesion, in contrast to what was observed in the other models, resulted in the total destruction of the vascular network, leading to a delay and an incomplete regeneration even one month post-injury.

Thus vasculature remodelling should be taken into account following transplantations where access to the blood milieu could impact on their phenotype and behaviour.

Notably, in the notexin-treated group, as soon as 12 days post-injury, multifocal calcification of necrotic myofibres appeared eliciting a peripheral granulomatous reaction with multinucleated giant cells.

These mineralised fibres remained even 6 months post-injury, as the chronic recruitment and activation of macrophages, even when the spared muscle tissue is completely regenerated.

The behaviour of resident or transplanted satellite cells would, for example, be influenced by the higher level of IL6 which would favour differentiation over self-renewal [ 45 — 47 ].

The muscle injury models examined here are extensively used in the literature to study tissue regeneration and stem cell properties, however our studies show that the nature of the model of injury should be chosen carefully depending on the experimental design and desired outcome. cytokine levels could have a major impact on muscle stem and stromal cell behaviour.

Freeze Injury : After skin incision A and muscle exposition B the Tibialis anterior was frozen with three consecutive cycles of freeze-thawing by applying for 15 sec a liquid nitrogen cooled metallic rod C, D.

The skin was then sutured E. A Satellite cell counts by flow cytometery Tg : Pax7nGFP mouse. C, D Haematoxylin and eosin stain of control, non-injured muscle. Scale bar represent 50 μm. E Control, uninjured TA muscle displaying vessel CD31, red and laminin green immunolabeling.

Scale bar represent 10 μm. A 3 months, B 6 months and C one month after freeze reinjury. D 3 months, E 6 months and F one month after NTX reinjury. G 3 months, H 6 months and I one month after CTX reinjury.

J 3 months, K 6 months and L one month after BaCl 2 reinjury. A 18h, B 4 days, C 12 days and D one month post freeze injury. E 18h, F 4 days, G 12 days and H one month post NTX injury. I 18h, J 4 days, K 12 days and L one month post CTX injury.

M 18h, N 4 days, O 12 days and P one month post BaCl 2 injury. A-C Haematoxylin and eosin stain control A , 18h B , and 1 month C , after aperture of the skin. D-F Sirius Red staining collagen deposits on control D , 18h E , and 1 month F , post open skin. G-I Immunohistochemistry of CD31 red and Laminin green in the freeze injury model, control G 18h H and 1 month I after aperture of the skin.

A 18h, B 4 days and C one month after CTX injury. D 18h, E 4 days and F one month after BaCl 2 injury. Vessel organisation after freeze injury 18h H and BaCl 2 injury 18h I. Scale bar represents 10 μm. J Vessel numbers per fibre 1 month after injury 50μL in all injury models.

K-M Percentage of remaining Pax7 positive cells K 18h, L 4 days and M 1 month post-injury on TA sections. N-P Percentage of activated Ki67 positive satellite cells N 18h, O 4 days and P 1 month after injury. A-D Haematoxylin and eosin stain 18h A ; 4 days, arrows indicate regeneration front B ; 12 days arrows indicate regeneration front C and 1 month D post-injury.

Scale bar represent 50 μm E-H Immunohistochemistry of CD31 red and Laminin green in the freeze injury model, 18h E , 4 days F , 12 days G and 1-month H post-injury.

Scale bar represent 50 μm I-L Images show blood vessel organisation in 3D 18h I , 4 days J , 12 days K , 1-month L post-injury. Scale bar represent 10 μm, arrows indicate anastomoses. M-P Count of the number of inflammatory cells per section 18h, 4 days, 12 days and 1 month post-injury.

Selected cytokines are displayed IL6 blue, IL10 green, IL12p40 yellow, IL12p70 red, MCP1 grey, MIP1a orange, MIP1b black. R number of satellite cells, counted by cytometry in one specific TA muscle in the control non-injured , 18h, 1 month, 3 month and 28 days after re-injury.

A-D Haematoxylin and eosin stain 18h A ; 4 days, B ; 12 days C and 1 month D post-injury. Scale bar represent 50 μm E-H Immunohistochemistry of CD31 red and Laminin green in the NTX injury model, 18h E , 4 days F , 12 days G and 1-month H post injury.

Scale bar represent 50 μm I-L Images show blood vessel organisation in 3D 18h I , 4 days J , 12 days K , 1-month L post-injury arrows indicate anastomoses. R number of satellite cells, counted by cytometry in one specific tibialis anterior muscle in the control non-injured , 18h, 1 month, 3 month and 28 days after re-injury.

Scale bar represent 50 μm E-H Immunohistochemistry of CD31 red and Laminin green in the CTX injury paradigm, 18h E , 4 days F , 12 days G and 1-month H post-injury.

Scale bar represent 50 μm I-L Images show blood vessel organisation in 3D 18h I , 4 days J , 12 days K , 1-month L post-injury; arrows indicate anastomoses. R number of satellite cells, counted by cytometry in one specific Tibialis anterior in the control non-injured , 18h, 1 month, 3 month and 28 days after re-injury.

Scale bar represent 50 μm E-H Immunohistochemistry of CD31 red and Laminin green in the BaCl 2 injury model, 18h E , 4 days F , 12 days G and 1-month H post injury.

R Number of satellite cells, counted by cytometry in one specific Tibialis anterior in the control non-injured , 18h, 1 month, 3 month and 28 days after re-injury. The authors also thank Patrick Ave, Patricia Flamant, Catherine Fitting, Huot Khun, Sabine Maurin for their excellent technical support.

Conceived and designed the experiments: DH AB ML GJ DB CT QP AG BGM JMC ST PR FC. Performed the experiments: DH AB ML DB CT QP FC GJ AG PR JMC. Analyzed the data: DH ML AB GJ PR FC QP. Wrote the paper: DH PR ST FC.

Statistical analysis: DH ML AB GJ PR. Morphometric analysis: DH PR GJ FC QP. Muscle injury: DH AB ML DB CT QP. Tissue preparation: DH DB AB. Histological analysis: DH FC GJ. Immunohistochemistry: DH DB AB QP. Satellite cell counting and cell sorting analysis: DH AG. Multiplex cytokine: DH.

Chemokine analysis: DH JMC. Contributed to the concepts design and coordination of all aspects of the experiments, interpretation of the data, and coordination of manuscript preparation and submission: PR ST FC. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.

Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Background A longstanding goal in regenerative medicine is to reconstitute functional tissus or organs after injury or disease. Results We compared the 4 most commonly used injury models i.

Conclusions Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Material and Methods Ethics All mice were housed in a level 2 biosafety animal facility, and received food and water ad libitum.

Muscle injury procedures Freeze Injury. Myotoxin injury. Chemical injury. Tissue preparation and histological analysis For histopathological analysis, right and left TA muscles were collected and snap-frozen in liquid nitrogen-cooled isopentane.

Download: PPT. Table 1. Summary and references of the primary antibodies used. Satellite cell counting To investigate satellite cell counts in one single isolated TA muscle we used transgenic Tg : Pax7nGFP mouse allowing the prospective selection by cytometry FACS and cell counting S2A Fig.

Statistical Analysis Data are expressed as mean±SEM, unless otherwise indicated. Morphometric analysis Two-dimension analysis was performed. Results The four injury models examined here were investigated using a variety of readouts, and the outcome of this analysis is documented in Table 1.

Fig 1. Number of satellite cells and their behaviour in the 4 different injury models. Differential loss of satellite cells in different injury models Freeze-injury.

BaCl 2. Altered histopathology following muscle trauma Skeletal muscle regeneration is a highly stereotypical process regardless of the injury method.

Fig 2. Muscle histology at different time points after injury. Fig 3. Fibre quantification and vascularization at different time points post injury in the 4 injury models. Table 2. Qualitative and semi-quantitative summary of the histological study for the four injury models at the different time points.

Inflammation We carried out a global analysis of inflammatory processes during muscle regeneration by investigating inflammatory cell infiltrates in parallel to cytokine and chemokine expression Fig 4 and Table 3. Table 3. Summary of the inflammatory infiltrate in the different injury models cell number per 10 microscopic fields.

Fig 4. Characterization of inflammation after injury in the 4 injury models. Remodelling of vasculature Freeze-injury. Fig 5. Three dimensional analysis of vessels at different time points in all 4 injury models.

Fibrosis Fibrosis, characterised by migration and proliferation of fibroblasts into the site of injury and excessive production of extra-cellular-matrix ECM proteins by these cells replacing normal tissue structure, is the hallmark of compromised or failed muscle regeneration.

Basal lamina Using immunofluorescence, we showed that although the expression and organisation of laminin 1, a major component of muscle basal lamina, was altered particularly in the FI model, a ghost of laminin 1 was consistently preserved around each muscle fibre 18h post-injury.

Discussion Skeletal muscle, skin and liver have been used as reference tissues for the study of tissue regeneration. Conclusion The muscle injury models examined here are extensively used in the literature to study tissue regeneration and stem cell properties, however our studies show that the nature of the model of injury should be chosen carefully depending on the experimental design and desired outcome.

Supporting Information. S1 Fig. Muscle freeze injury procedure. s TIF. S2 Fig. Characterization of normal muscle satellite cells, histology, basal lamina and vessels.

S3 Fig. Histological study 3 and 6 months after injury and reinjury. S4 Fig. Characterization of vessels and basal lamina after injury in the 4 injury models. S5 Fig. Aperture of the skin sham. S6 Fig. Injury models 50 μL injection. S7 Fig. Summary of the freeze injury model regeneration process.

S8 Fig. Summary of NTX injury model regeneration process. S9 Fig. Summary of CTX injury model regeneration process. S10 Fig. Summary of BaCl2 injury model regeneration process. Acknowledgments The authors also thank Patrick Ave, Patricia Flamant, Catherine Fitting, Huot Khun, Sabine Maurin for their excellent technical support.

Author Contributions Conceived and designed the experiments: DH AB ML GJ DB CT QP AG BGM JMC ST PR FC. References 1. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. Gayraud-Morel B, Chrétien F, Tajbakhsh S. Skeletal muscle as a paradigm for regenerative biology and medicine.

Regen Med. Tajbakhsh S. Skeletal muscle stem cells in developmental versus regenerative myogenesis. Journal of Internal Medicine. Zammit PS, Partridge TA, Yablonka-Reuveni Z.

The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem. Arnold L, Henry A, Poron F, Baba-Amer Y, van Rooijen N, Plonquet A, et al. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.

J Exp Med. Abou-Khalil R, Mounier R, Chazaud B. Cell Cycle. Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Exercising these muscles can help to restore muscle function and minimize functional impairments.

Age-related muscle loss is also a target of physical therapy, as exercise can reduce the effects of age-related atrophy and improve muscle function. The goal of a physiotherapist is to improve physical functioning and reduce functional impairments; this is achieved by understanding the cause of muscle impairment and assessing the capabilities of a patient, after which a program to enhance these capabilities is designed.

Some factors that are assessed include strength, balance, and endurance, which are continually monitored as exercises are introduced to track improvements in muscle function. Physiotherapists can also instruct patients on the proper use of equipment, such as crutches, and assess whether someone has sufficient strength to use the equipment and when they can function without it.

Muscle tissue arises from embryonic mesoderm. Somites give rise to myoblasts and fuse to form a myotube. The nucleus of each contributing myoblast remains intact in the mature skeletal muscle cell, resulting in a mature, multinucleate cell.

Satellite cells help to repair skeletal muscle cells. Smooth muscle tissue can regenerate from stem cells called pericytes, whereas dead cardiac muscle tissue is replaced by scar tissue. Aging causes muscle mass to decrease and be replaced by noncontractile connective tissue and adipose tissue.

Why is muscle that has sustained significant damage unable to produce the same amount of power as it could before being damaged?

If the damage exceeds what can be repaired by satellite cells, the damaged tissue is replaced by scar tissue, which cannot contract.

Explain your answer. Smooth muscle tissue can regenerate from stem cells called pericytes, cells found in some small blood vessels.

These allow smooth muscle cells to regenerate and repair much more readily than skeletal and cardiac muscle tissue. Development and Regeneration of Muscle Tissue Copyright © by OpenStaxCollege is licensed under a Creative Commons Attribution 4.

Skip to content Muscle Tissue. Learning Objectives By the end of this section, you will be able to: Describe the function of satellite cells Define fibrosis Explain which muscle has the greatest regeneration ability.

Career Connections. Chapter Review Muscle tissue arises from embryonic mesoderm.

Muscle regeneration process -

Together, our study provides valuable methods that are useful in evaluating muscle regeneration and the efficacy of therapeutic strategies for muscle diseases. Skeletal muscle consists mainly of myofibers, which are large cylindrical cells with many nuclei. Myofibers are terminally differentiated post-mitotic cells; however, skeletal muscles possess a high ability to regenerate.

Regeneration of mature myofibers is dependent on satellite cells. Satellite cells are mononucleated cells located between the plasma membrane of the myofiber and basal lamina. They normally remain in a quiescent state, but are activated upon muscle injury, and then they proliferate and differentiate to regenerate myofibers.

Genetically engineered mice in which satellite cells are ablated show a complete lack of regenerative response Lepper et al.

Furthermore, single-satellite cell transplantation revealed that these cells indeed possess self-renewal potential, in addition to the ability to differentiate into myofibers Sacco et al.

Thus, satellite cells are considered as definitive adult muscle stem cells. Adult myogenesis is a highly ordered process in which satellite cells proliferate, differentiate, and generate new myofibers.

Myogenic regulatory factors MRFs are important regulators of myogenesis and their expression is tightly regulated. Quiescent satellite cells do not express detectable levels of MyoD but they begin to express high levels of MyoD upon activation Zammit et al.

Expression of MyoD is maintained during the proliferation phase and continues until the early differentiation phase Zammit et al.

Myogenin is not expressed in quiescent satellite cells and proliferating undifferentiated myoblasts, but its expression is significantly upregulated when cells begin to differentiate Bentzinger et al. Therefore, MyoD and Myogenin are commonly used as activation and differentiation markers of myogenesis, respectively.

Expression levels of MRF4 are highest of the MRFs in adult mature muscle and are considered to reflect muscle fiber maturity Bentzinger et al.

Adult muscle regeneration recapitulates many aspects of embryonic myogenesis, including expression of embryonic- or perinatal-type myosin heavy chain MyHC Sartore et al.

Thus, expression of these embryonic-type contractile proteins is a hallmark of muscle regeneration and is often used to detect activity of regeneration. Expression of MRFs or embryonic or perinatal MyHC is useful to examine regenerating events.

However, the most important goal in tissue regeneration is that the normal condition is restored. From this perspective, expression of the above described regeneration markers reflects conditions where muscle is still abnormal. If diseased muscle is successfully treated and restored to its healthy state, expression of regeneration markers should be downregulated.

Based on this notion, some studies examined downregulation of embryonic MyHC to assess therapeutic efficacy Guiraud et al.

However, little is known about indicators that directly reflect normality of muscle tissue. Although experimental muscle regeneration is a highly ordered process, it is not completely synchronized, and thus there is a regional difference in the progression of regeneration within a single muscle.

In a regeneration model of grafted muscle, it was reported that a radial gradient of regeneration is formed, with more mature muscle at the periphery and less mature muscle toward the center in the regenerating grafted muscle Carlson and Gutmann, Likewise, other muscle regeneration models, including cardiotoxin injury models, do not show completely uniform regeneration, with some regions showing accelerated regeneration while other regions are in a delayed phase of regeneration.

Therefore, it is important to develop a reliable method for evaluating muscle regeneration accurately and quantitatively, taking spatial non-uniformity of regeneration into account. In this study, we carefully examined several regeneration-related markers during muscle regeneration.

These analyses revealed that expression of Myozenin Myoz1 and Myoz3 , Troponin I Tnni2 , and Dystrophin Dmd correlates very well with the progression of regeneration. Their expression highly reflects myofiber maturity because high expression of these genes can only be achieved in muscle tissue in vivo and not in cultured myotubes in vitro.

We also developed a method that can distinguish advanced regenerating areas from delayed regenerating areas within single muscle, which enables accurate and quantitative evaluation of muscle regeneration.

Our study provides useful information for the studies of muscle regeneration and therapy for muscle diseases. All animal experiments performed in this report were approved by the Animal Care and Use Committee of Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.

Cardiotoxin CTX, Sigma was dissolved in sterile saline at a concentration of 10 μM. Tibialis anterior TA muscles of 2 to 3 month old mice were injected with μl CTX.

TA muscles were isolated at days 0, 3, 5, 7, and 14 of CTX injury, embedded in tragacanth gum, and frozen in liquid nitrogen-cooled isopentane. Isolation of mouse satellite cells was reported previously Uezumi et al.

Hind limb muscles were collected, minced and digested with 0. Digested muscles were passed through an gauge needle several times and further digested for 30 min at 37°C.

Digested samples were filtered through a μm cell strainer, and then through a μm cell strainer. Total RNA was extracted from cultured satellite cells and muscles using RNeasy Mini Kit Qiagen and miRNeasy Mini Kit Qiagen , respectively.

Pieces of muscle tissues collected from frozen TA muscles were crushed in QIAzol Lysis Reagent Qiagen using a Shakeman homogenizer Bio Medical Science.

Complementary DNA cDNA was synthesized using QuantiTect Transcription Kit Qiagen. qRT-PCR was performed with SYBR Premix Ex Taq II Takara on a Takara Thermal Cycler Dice Real Time System Takara under the following cycling conditions: 94°C for 30 s followed by 40 cycles of amplification 94°C for 5 s, 60°C for 20 s, 72°C for 12 s and dissociation curve analysis.

For gene expression analysis in regenerating TA muscles and differentiating satellite cells, mRNA expression was normalized with Cmas.

Relative mRNA expression was then calculated using the 2 —ΔΔ method. Specific primers used for qRT-PCR were listed in Supplementary Table 1. Primers for Actb were provided from QuantiTect Primer Assays Kit Qiagen.

Frozen transverse sections were cut at the thickness of 8 μm and fixed for 5 min in ice-cooled acetone. After blocking with M. TM mouse IgG blocking reagent Vector Laboratories , sections were incubated overnight at 4°C with primary antibodies diluted in M.

TM diluent. After washing with PBS, sections were stained with secondary antibodies. Primary and secondary antibodies used were listed in Supplementary Table 2. Nuclei were counterstained with DAPI Dojindo , and stained muscles were mounted with SlowFade Diamond anti-fade reagent Invitrogen.

Fluorescent signals were detected with confocal laser scanning microscope systems TCS-SP8 Leica. The same sections were stained with hematoxylin and eosin HE after capturing fluorescent images.

HE images were taken with microscope AXIO Carl Zeiss equipped with a digital camera, Axiocam ERc 5s Carl Zeiss. Cross-sections were made by cutting at the mid-belly of TA muscle at the position about 3 mm from proximal end of TA muscle.

After immunostaining, fluorescent images of entire cross-sections were captured with fluorescent microscope system BZ-X Keyence. Image recognition and quantification were performed by using the Hybrid Cell Count Application Keyence. First, entire cross-sectional areas of TA muscle were measured.

For quantification of Myoz1-positive area, Myoz1-stained area was recognized based on the intensity of Myoz1 staining by adjusting threshold. For quantification of dystrophin-positive area, dystrophin-stained sarcolemma was first recognized based on the intensity of dystrophin staining by adjusting threshold, and then dystrophin-positive fiber area was recognized by using inversion function.

After recognition of Myoz1- and dystrophin-positive areas, the misrecognized small areas were excluded by adjusting lower limit in histogram function. Finally, errors in recognition step were corrected manually, and then Myoz1- and dystrophin-positive areas were measured.

Myoz1- or dystrophin-positive area was divided by entire cross-sectional area to calculate percentage of area positive for each marker. Two side unpaired t -test was used to compare two groups.

Statistical significance was evaluated using GraphPad Prism 8. We first analyzed the expression of several internal control genes by qRT-PCR to determine the optimum control genes for the most accurate gene expression analysis during muscle regeneration.

As shown in Figure 1 , Gapdh and Actb also called β-actin , commonly used control genes, were highly variable in their expression during muscle regeneration Figure 1.

Therefore, these genes are not suitable as internal control genes to normalize expression of target genes. One pioneering study on comprehensive gene expression analysis during muscle regeneration had previously pointed out this problem and identified two genes that are stably expressed across all time points during muscle regeneration Zhao and Hoffman, Those two genes are Cmas also called as CMP-N-acetylneuraminic acid synthase and Eif3c called as NIPI-like protein.

We thus examined the expression of Cmas and found relatively stable expression of this gene during muscle regeneration Figure 1. Therefore, we decided to use Cmas as an internal control gene for gene expression analysis during muscle regeneration.

Figure 1. Optimum internal control genes for gene expression analysis during muscle regeneration. A Amplification curves of quantitative reverse transcription-PCR qRT-PCR for Gapdh , β -actin Actb , and Cmas using total RNA extracted from intact and regenerating tibialis anterior TA muscles 3, 5, 7, and 14 days after CTX injury.

Coefficient of variation CV is shown in the graphs. Note that Ct value of Cmas showed smaller CV than that of Gapdh or Actb. We next examined expression of several regeneration-related genes. As expected, expression of MyoD and Myogenin were highly induced upon muscle injury, and gradually downregulated thereafter Figure 2A.

We also observed similar dynamics in the expression of embryonic-type contractile genes. As shown in Figure 2B , expression of Myh3 and Myh8 was detected at day 3 of muscle injury, reached its peak at day 5, and then decreased to levels comparable to intact muscle. Thus, expression of above-described genes is transient during muscle regeneration and therefore does not reflect completion of regeneration accurately.

Zhao et al. Those include Myozenin , which encodes a Z-disk associated protein myozenin, and Tnni2 , which encodes a fast skeletal type troponin I, a protein responsible for the calcium-dependent regulation of muscle contraction.

Therefore, we examined expression of these muscle structural component genes. Expression of Myoz1 , Myoz3 , and Tnni2 was sharply downregulated at day 3 of muscle injury, and then gradually upregulated as regeneration proceeded Figure 2C , indicating that expression of these genes well reflects the extent of muscle regeneration.

We also analyzed expression of Dmd , which encodes a dystrophin protein, and Myh4 , which encodes a MyHC-IIb, a predominant type of MyHC expressed in TA muscle Kammoun et al.

Various strategies, including growth factors injections, transplantation of muscle stem cells in combination or not with biological scaffolds, anti-fibrotic therapies and mechanical stimulation, may become therapeutic alternatives to improve functional muscle recovery.

Satellite cells SC are skeletal muscle stem cell located between the plasma membrane of myofibers and the basal lamina. Their regenerative capabilities are essential to repair skeletal muscle after injury Hurme and Kalimo ; Lipton and Schultz Sambasivan et al.

After injury, SC become activated, proliferate and give rise to myogenic precursor cells, known as myoblasts. After entering the differentiation process, myoblasts form new myotubes or fuse with damaged myofibers, ultimately mature in functional myofibers.

Skeletal muscle injuries can stem from a variety of events, including direct trauma such as muscle lacerations and contusions, indirect insults such as strains and also from degenerative diseases such as muscular dystrophies Huard et al. Skeletal muscle can regenerate completely and spontaneously in response to minor injuries, such as strain.

In contrast, after severe injuries, muscle healing is incomplete, often resulting in the formation of fibrotic tissue that impairs muscle function. Although researchers have extensively investigated various approaches to improve muscle healing, there is still no gold standard treatment.

This concise review provides a sight about the various phases of muscle repair and regeneration, namely degeneration, inflammation, regeneration, remodeling and maturation. We also address the therapeutic potential of mechanical stimulation and of anti-fibrotic therapy to enhance muscle regeneration and repair.

Skeletal muscle has a robust innate capability for repair after injury through the presence of adult muscle stem cells known as satellite cells SC. The disruption of muscle tissue homeostasis, caused by injury, generates sequential involvement of various players around three main phases Fig.

Sequential cycle of muscle healing phases after laceration. Histological images adapted from Menetrey et al, Am J Sports Med sp: superficial portion, de: deepest part.

Active muscle degeneration and inflammation occur within the first few days after injury. The initial event is necrosis of the muscle fibers, which is triggered by disruption of local homeostasis and particularly by unregulated influx of calcium through sarcolemma lesions Tidball Excess in cytoplasmic calcium causes proteases and hydrolases activation that contribute to muscle damage and also causes activation of enzymes that drive the production of mitogenic substances for muscle and immune cells Tidball After muscle degeneration, neutrophils are the first inflammatory cells infiltrating the lesion.

A large number of pro-inflammatory molecules such as cytokines TNF-α, IL-6 , chemokine CCL17, CCL2 and growth factors FGF, HGF, IGF-I, VEGF; TGF-β1 are secreted by neutrophils in order to create a chemoattractive microenvironment for other inflammatory cells such as monocytes and macrophages Tidball ; Toumi and Best Two types of macrophages are identified during muscle regeneration McLennan , which appear sequentially during muscle repair Arnold et al.

M1 macrophages, defined as pro-inflammatory macrophages, act during the first few days after injury,. contribute to cell lysis, removal of cellular debris and stimulate myoblast proliferation. Conversely, M2 macrophages, defined as anti-inflammatory macrophages, act 2 to 4 days after injury, attenuate the inflammatory response and favor muscle repair by promoting myotubes formation Tidball and Wehling-Henricks ; Chazaud ; Chazaud et al.

Macrophages, infiltrating injured muscle, are key players of the healing process Zhao et al. Muscle regeneration usually starts during the first 4—5 days after injury, peaks at 2 weeks, and then gradually diminishes 3 to 4 weeks after injury.

A fine balance between these mechanisms is essential for a full recovery of the contractile muscle function. Muscle fibers are post-mitotic cells, which do not have the capacity to divide. Following activation, SC proliferate and generate a population of myoblasts that can either differentiate to repair damaged fibers or, for a small proportion, self-renew to maintain the SC pool for possible future demands of muscle regeneration Collins ; Dhawan and Rando SC cycle progression and cell fate determination are control by complex regulatory mechanisms in which, intrinsic and extrinsic factors are involved Dumont et al.

Connective tissue remodeling is an important step of the regenerative muscle process. Rapidly after muscle injury, a gap is formed between damaged muscle fibers and filled with a hematoma. Muscle injuries can be clinically classified depending of the nature of the hematoma size, location. Late elimination of the hematoma is known to delay skeletal muscle regeneration, to improve fibrosis and to reduce biomechanical properties of the healing muscle Beiner et al.

In rare complication, major muscle injuries may lead to the development of myositis ossificans that will impair muscle regeneration and repair Beiner and Jokl Walczak et al.

The presence of fibrin and fibronectin at the injury site, initiate the formation of an extracellular matrix that is rapidly invaded by fibroblasts Darby et al. In its initial phase, the fibrotic response is beneficial, stabilizing the tissue and acting as a scaffold for myofibers regeneration.

Nevertheless, an excessive collagen synthesis post injury, often result in an increase of scar tissue size over time that can prevent normal muscle function Mann et al. Many growth factors are involved in the development of fibrosis, such as Connective Tissue Growth Factor CTGF , Platelet-Derived Growth Factor PDGF or myostatin.

Although fibroblasts are the major collagen-producing cells in skeletal muscle, TGF-β1 have also an effect directly on myoblasts causing their conversion to myofibroblasts.

Thus myoblasts initially acting to repair damaged myofibers, will produce significant level of collagen and will contribute to muscle fibrosis Li and Huard The restoration of the blood supply in the injured skeletal muscle is one of the first signs of muscle regeneration and is essential to its success.

Without revascularization, muscle regeneration is incomplete and a significant fibrosis occurs Best et al. After muscle trauma, blood vessels rupture induces tissue hypoxia at the injury site Jarvinen et al. New capillaries formation quickly after injury is therefore necessary Scholz et al. Secretion of angiogenic factors such as vascular endothelial growth factor VEGF at the lesion site is important and several studies have shown that VEGF, by favoring angiogenesis, improve skeletal muscle repair Deasy et al.

Muscle repair is complete when injured myofibers are fully regenerated and become innervated. The synaptic contact between a motor neuron and its target muscle fiber, often take place at a specific site in the central region of myofibers, the neuromuscular junction NMJ Wu et al.

NMJ are essential for maturation and functional activity of regenerating muscles. Within 2—3 weeks after muscle damage, the presence of newly formed NMJ is observed in regenerative muscle Rantanen et al.

Growth factors play a variety of roles in the different stages of muscle regeneration Grounds ; Menetrey et al. These biologically active molecules, synthetized by the injured tissue or by other cell types present at the inflammatory site, are release in the extracellular space and modulate the regenerative response Table 1.

Although hepatocyte growth factor HGF , fibroblast growth factor FGF and platelet-derived growth factor PDGF are of interest because of their capacity to stimulate satellite cells Sheehan et al. IGF-I stimulates myoblasts proliferation and differentiation Engert et al.

In a mouse model, direct injections of human recombinant IGF-I at two, five, and seven days after injury enhanced muscle healing in lacerated, contused, and strain-injured muscles Menetrey et al. However, the efficacy of direct injection of recombinant proteins is limited by the high concentration of the factor typically required to elicit a measurable effect.

Gene therapy may be an effective method by which to deliver high, maintainable concentrations of growth factor to injured muscle Barton-Davis et al. Although IGF-I improved muscle healing, histology of the injected muscle revealed fibrosis within the lacerated site, despite high level of IGF-I production Lee et al.

Another growth factor, VEGF, by favoring angiogenesis, is known to enhance skeletal muscle repair Deasy et al. By targeting simultaneously angiogenesis and myogenesis, it was shown that combined delivery of VEGF and IGF-I enhance muscle regenerative process Borselli et al.

In this direction, the use of platelet-rich plasma PRP is considered as a possible alternative approach based on the ability of autologous growth factors to improve skeletal muscle regeneration Hamid et al. Considered as safe products, autologous PRP injections are increasingly used in patients with sports-related injuries Engebretsen et al.

Nevertheless, a recent randomized clinical trial show no significant positive effects of PRP injections, as compared with placebo injections, in patients with muscle injuries, up to one year after injections Reurink et al. Customization of PRP preparation, as recently demonstrated by the use of TGF-β1 neutralizing antibodies, is a promising alternative to promote muscle regeneration while significantly reducing fibrosis Li et al.

Transplantation of satellite cell-derived myoblasts has long been explored as a promising approach for treatment of skeletal muscle disorders.

After an initial demonstration that normal myoblasts can restore dystrophin expression in mdx mice Partridge et al. Even recently, despite clear improvement in methodologies that enhance the success of myoblast transplantation in Duchenne patients Skuk et al.

These experiments have raised concerns about the limited migratory and proliferative capacities of human myoblasts, as well as their limited life span in vivo.

It led to the investigations of other muscle stem cells sources that could overcome these limitations and outperform the success of muscle cell transplantation. The use of such myogenic progenitors cells for improving muscle healing may become an interesting therapeutic alternative Tedesco and Cossu ; Tedesco et al.

Myogenic precursor cell survival and migration is greatly increased by using appropriate scaffold composition and growth factor delivery Hill et al. Controlling the microenvironment of injected myogenic cells using biological scaffolds enhance muscle regeneration Borselli et al. Ideally, using an appropriate extracellular matrix ECM composition and stiffness, scaffolds should best replicate the in vivo milieu and mechanical microenvironment Gilbert et al.

A combination of stem cells, biomaterial-based scaffolds and growth factors may provide a therapeutic option to improve regeneration of injured skeletal muscles Jeon and Elisseeff TGF-β1 is expressed at high levels and plays an important role in the fibrotic cascade that occurs after the onset of muscle injury Bernasconi et al.

Therefore, neutralization of TGF-β1 expression in injured skeletal muscle should inhibit the formation of scar tissue. Indeed, the use of anti-fibrotic agents ie decorin, relaxin, antibody against TGF-β1… that inactivate TGF-β1 signaling pathways reduces muscle fibrosis and, consequently, improve muscle healing, leading to a near complete recovery of lacerated muscle Fukushima et al.

Losartan, an angiotensin II receptor antagonist, neutralize the effect of TGF-β1 and reduce fibrosis, making it the treatment of choice, since it already has FDA approval to be used clinically Bedair et al.

Suramin, also approved by the FDA, blocks TGF-β1 pathway and reduces muscle fibrosis in experimental model Chan et al. Mechanical stimulation may offer a simple and effective approach to enhance skeletal muscle regeneration.

Stretch activation, mechanical conditioning but also massage therapy or physical manipulation of injured skeletal muscles have shown multiple benefit effects on muscle biology and function in vitro and in vivo Tatsumi et al.

Recently, Cezar and colleagues demonstrates that mechanical forces are as important biological regulators as chemicals and genes, and underlines the immense potential of developing mechano-therapies to treat muscle damage Cezar et al.

A recent study also demonstrated that a treatment based on ultrasound-guided intra-tissue percutaneous electrolysis EPI technique enhances the treatment of muscle injuries Abat et al. Altogether, these results suggest that mechanical stimulation should be considered as a possible therapy to improve muscle regeneration and repair.

Skeletal muscle injuries are very frequently present in sports medicine and pose challenging problems in traumatology. Despite their clinical importance, the optimal rehabilitation strategies for treating these injuries are not well defined.

After a trauma, skeletal muscles have the capacity to regenerate and repair in a complex and well-coordinated response. This process required the presence of diverse cell populations, up and down-regulation of various gene expressions and participation of multiples growth factors.

Strategies based on the combination of stem cells, growth factors and biological scaffolds have already shown promising results in animal models. A better understanding of the cellular and molecular pathways as well as a better definition of the interactions cell-cell and cell-matrix that are essential for effective muscle regeneration, should contribute to the development of new therapies in humans.

In this direction, a recent paper from Sadtler et al demonstrated that specific biological scaffold implanted in injured mice muscles trigger a pro-regenerative immune response that stimulate skeletal muscle repair Sadtler et al.

CTGF, connective tissue growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF-I, insulin like growth factor-I; NMJ, neuromuscular junction; PDGF, platelet derived growth factor; PRP, platelet rich plasma; SC, satellite cells; TGF-β1, transforming growth factor β1; VEGF, vascular endothelial growth factor.

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J Appl Physiol 95 2 — Charge SB, Rudnicki MA Cellular and molecular regulation of muscle regeneration. Conflicting results have also suggested cryotherapy could delay and impair the regeneration process. There are no definitive findings about the effects of cryotherapy on the process of muscle regeneration.

The aim of the present study was to evaluate the effects of a clinical-like cryotherapy on inflammation, regeneration and extracellular matrix ECM remodeling on the Tibialis anterior TA muscle of rats 3, 7 and 14 days post-injury.

Cryotherapy did not alter regeneration markers such as injury area, desmin and Myod expression. Despite regulating Collagen I and III and their growth factors, cryotherapy did not alter collagen deposition. In summary, clinical-like cryotherapy reduces the inflammatory process through the decrease of macrophage infiltration and the accumulation of the inflammatory key markers without influencing muscle injury area and ECM remodeling.

Skeletal muscle lesions are responsible for the majority of the functional limitations observed in sports and occupational medicine 1. After primary injury, muscle regeneration occurs in a highly orchestrated process that involves the activation of muscle satellite cells to proliferate and differentiate into a new muscle fiber 2 with a constant pattern irrespective of the cause contusion, strain, or laceration.

After muscle injury it is possible to observe four independent phases, despite their etiology: degeneration, inflammation, regeneration and fibrosis 2 , 3 , 4.

The activation and differentiation of satellite cells is characterized by the rapid upregulation of myogenic differentiation 1 MyoD and insulin-like growth factor 1 IGF-1 5 , 6. In addition, in vitro and in vivo studies indicate that anti-inflammatories such as interleukin IL and transforming growth factor beta TGF-β and pro-inflammatory cytokines such as tumor necrosis factor alpha TNF-α and nuclear factor- κB NF-κB produced by macrophages could activate satellite cells, stimulating myoblast proliferation and differentiation into myotube formation 7 , 8.

The fibrosis and remodeling phases of muscle regeneration involve the deposition of Collagen I and III fibers and reorganization of the tissue, which could be induced by TGF-β 9 , IGF-I 10 and connective tissue growth factor CTGF In addition, matrix metalloproteinases MMPs cooperatively degrade all components of the extracellular matrix ECM MMP-2 or gelatinase A activity is concurrent with the regeneration of new myofibers probably due to degradation of type IV collagen of the basement membrane during myoblast proliferation, migration and fusion.

MMP-9 or gelatinase B activation is related to the early inflammatory phase and to the activation of satellite cells 13 , The primary role of cryotherapy post-injury in sports medicine is to reduce pain, swelling, degeneration and inflammation 2 , 15 , 16 , Some studies have demonstrated that cryotherapy minimizes secondary hypoxic injury through the reduction of cellular metabolism and injury area 17 , 20 , 21 , 22 , 23 , Another study demonstrated that cryotherapy for six consecutive hours reduced macrophage infiltration, edema formation and myonecrosis desmin ratio of rat muscle However, excessive or prolonged cooling would be damaging in clinical practice On the other hand, previous studies suggest that cryotherapy, despite decreasing inflammation 24 , 26 , might delay the muscle regeneration process 4.

Cryotherapy could retarded the migration of macrophages in the injured area and the secretion of growth factors such as TGF-β1 and IGF-I expression, which might be harmful for muscle regeneration 4. They speculated that macrophages play important roles not only for degeneration, but also for muscle regeneration and therefore the influence of cryotherapy on macrophage activities might be closely related to a delay in muscle regeneration, impairment of muscle regeneration and redundant collagen synthesis 4.

Collectively, few studies have evaluated the effects of cryotherapy on muscle regeneration with intermittent and clinical-like protocols related to those used in humans 4 , 22 , Our hypothesis was that clinical-like cryotherapy, applied to the tibialis anterior TA muscle immediately after injury, would minimize the inflammatory process, secondary injury area and harmful collagen adaptation.

Therefore, the aim of this study was to evaluate the effects of cryotherapy on inflammation TNF-α, macrophages, NF-κB, TGF-β and MMP-9 , regeneration MyoD, IGF-1 and desmin , the extracellular matrix Collagen I and III, CTFG and MMP-2 , the injury area and muscle morphology of rat TA post-injury.

This study was divided into 3 experimental analysis periods: 3, 7 and 14 days after muscle lesion. To induce muscle injury in the middle belly of the right TA, the skin around the muscle was trichotomized and cleaned. After that, the skin was sutured 27 , This model induces a homogeny injury area and restricts the surface region of muscle belly 22 , 27 , 29 , which is similar to mechanism of muscle contusion model 2.

Moreover, it is a model of easy applicability that allows a good reproducibility of experiment and less variability in the extension of muscle damage among animals 22 , 27 , Under anesthesia, cryotherapy was performed immediately after muscle injury.

The animals were maintained in a horizontal position on a plastic table and the ankle of right hindlimb was maintained by tape for the exposition of the TA muscle skin. The sessions of cryotherapy consisted of the application of a plastic pack filled with crushed ice, maintained by the tape directly on the skin of the right TA muscle 22 , After the experimental periods, the animals were anesthetized and weighed.

Then, the right TA muscles were carefully removed and weighed. The muscles were then divided into two parts at the middle of the belly: the proximal fragment was used for the histological and immunofluorescence analysis and the distal one for the mRNA analysis.

One image of all muscle cross-sections was acquired at low magnification and centralized nuclei were counted as a percentage of total muscle fibers. Since the primary injury was standardized for all damaged muscles, possible differences in the final area of injury were considered because of different extensions in the secondary muscle injury.

The qualitative analysis of histological sections stained with Toluidine Blue included a description of the stages of tissue repair, involving the presence and type of inflammatory infiltrate, edema, necrosis and immature fibers of all experimental groups 22 , 27 , The muscle preparation for immunofluorescence assay was previous described The primary antibody used for immunostaining was: a rabbit Desmin dilution , catalog no.

AB Abcam, Cambridge, MA and the secondary antibody was rhodamine red goat anti-rabbit IgG dilution , catalog no. Rb; Molecular Probes, Eugene, OR ; b rat laminin dilution , catalog no. AB Abcam, Cambridge, MA and the secondary antibody was Alexa-Fluor IgG anti-mouse dilution , catalog no.

A Molecular Probes, Eugene, OR ; c mouse anti rat CD68 dilution , catalog no. MCAR ABD Serotec, Kidlington and the secondary antibody was Alexa-Fluor IgG anti-mouse dilution , catalog no. A Molecular Probes, Eugene, OR ; d rabbit anti-TNFα dilution , catalog no. NBP Novus Biologicals, Littleton, CO and the secondary antibody was rhodamine red goat anti-rabbit IgG dilution , catalog no.

Rb Molecular Probes, Eugene, OR ; e mouse Anti-Collagen, Type III dilution , catalog no. C Sigma-Aldrich, St. Louis, MO and the secondary antibody was Alexa-Fluor IgG anti-mouse dilution , catalog no.

A Molecular Probes, Eugene, OR ; f mouse Anti-Collagen, Type I dilution , catalog no. A Molecular Probes, Eugene, OR. For quantitative measurements of immunoreactivity, images of five different regions from the middle-belly of the TA muscles were captured Axiocam, Carl Zeiss, Jena, Germany at a final magnification of 20×, with the microscopic setting kept the same for all slides.

Regions categorized as degenerative were those which included fibers with hypercontraction, delta lesion, vacuolated and ghost cells, whereas regenerative regions included myoblasts, myotubes and central-nucleate myofibers.

In both, the area occupied by fibers positive for the CD68 and TNF-α-labeled antibodies was determined by computer aided image analysis Image-Pro Express software, Media Cybernetics, Silver Spring, MD, USA and calculated as percentage. The percentage of CD68 and TNF-α-labeled area degenerative or regenerative ones was assessed by multiplying them by and dividing them by the total number of TA muscle fibers.

The interstitial space was not considered when the degenerated and regenerated areas were calculated Quantification of Collagen I and Collagen III was performed by ImageJ software using the tool color histogram version 1.

The extracted RNA was dissolved in hydroxymethyl-aminomethane·hydrochloride tris-HCl and ethylenediaminetetracetic acid TE , pH 7.

The integrity of the RNA was confirmed by inspection of ethidium bromide stained 18S and 28S ribosomal RNA under violet ultra-light.

Total RNA was reverse transcribed into complementary deoxyribonucleic acid cDNA as previous described For each gene, all samples were amplified simultaneously in duplicate in one assay run. The GAPDH mRNA was used as internal control 27 , Statistical analysis was performed using the Statistica 7.

Cryotherapy groups showed a linear decrease in the surface temperature of the TA muscle during its application. On average, the temperature decreased by The temperature of the control group decreased 1. Alterations of the temperatures at the surface of the tibialis anterior TA muscle during cryotherapy.

Vertical axis shows the temperature in degree Celsius. Horizontal axis shows the time minutes. Cross-sections of TA muscle evaluated 3 days after cryolesion showed several stages of myonecrosis: presence of necrotic muscle fibers, intense presence of cellular infiltration and clear areas among the muscle fibers Fig.

As expected, the 7 days-after-injury group showed fewer inflammatory signs, observed by a decrease in cellular infiltration. In this period, it is also possible to note an intense regeneration process through the presence of many small fibers with centralized nuclei, as well as the presence of a large nucleus and prominent nucleolus in basophilic fibers featuring ribosomal activity Fig.

Regeneration fibers with basophilic and centralized nucleus fibers after 14 days of cryolesion were also noted. In this time, fibers with similar morphology compared to control group were also observed Fig.

Interestingly, cryotherapy had decreased cellular infiltration 3 and 7 days after injury Fig. There is no difference in the cellular infiltration of the cryotherapy group after 14 days of muscle injury Fig. Cryotherapy did not alter morphological aspects of the regeneration process in any evaluated groups Fig.

a control group without injury. b signs of muscle tissue damage were identified 3 days after cryolesion by presence of necrotic muscle fibers NF and intense presence of cellular infiltration asterisks. d note presence of small muscle fibers in intense regeneration process with centralized nucleus head arrows and basophilic fibers showing prominent nucleolus , also note presence of cellular infiltration asterisks 7 days after cryolesion, however with less intensity compared to 3 days.

f muscle fibers in regeneration process 14 days after cryolesion, but still is possible note presence of centralized nuclei fibers head arrows , basophilic fibers and a minimum presence of cellular infiltration asterisk compared with 3 and 7 days after injury.

These data demonstrate cryotherapy did not modify regulation of MyoD observed 3 days after injury. The mRNA levels of MyoD, NF-κB, TNF-α, MMP-9, MMP-2, TGF-β, CTGF, IGF-1, Collagen I and Collagen III of Tibialis anterior TA muscle.

IGF-1 mRNA levels were positively increased only at 3 days post-lesion compared to the control group L3: However, IGF-1 mRNA levels did not change for other periods of treatment compared with the same period without treatment.

In the muscle injury group NF-κB mRNA levels increased at 3, 7 and 14 days post-lesion compared to control group L3: 7. The L3 group showed increased mRNA levels of TNF-α L3: 5. Collagen I mRNA levels increased in the L7 group compared to the control group L7: The muscle injury group increased Collagen III mRNA levels at 3 and 7 days compared to the control group L3: 98 fold; L7: Only the L3 group increased CTGF mRNA levels compared to the control group L3: Cryotherapy groups did not alter MMP-2 mRNA levels compared to injury muscles in all evaluated periods Fig.

Muscle injury increased MMP-9 mRNA levels only at 3 days post-lesion compared to control group L3: 5. It was noted in both 7 and 14 days post-lesion, as well as for cryotherapy in the same periods, that mRNA levels of MMP-9 returned to baseline values, similar to control Fig.

It was also possible to note presence of desmin negative fibers in some fibers, possibly in the process of regeneration. The negative control showed no staining.

Desmin immunofluorescence in regenerating tibialis anterior TA muscles. Frozen muscle sections were immunostained for desmin red , Laminin green and nuclei blue.

Micrographs are representative of muscle cross sections observed on day 3 after muscle damage L3 group a and days 7 after muscle damage L7 group b. Serial muscle sections stained for desmin indicate desmin-positive fibers arrow and the bundle organization of myofibers was not preserved in injury area asterisk.

Frozen muscle sections were immunostained for CD68 green , desmin red and nuclei blue. Cryotherapy treatment reduced the percentage of muscle fibers infiltrated by macrophages in 3 and 7 days post-lesion. It was also noted that cryotherapy prevented TNF-α from infiltrating muscle fibers as well as the injured area Fig.

C figure are computer-generated merged image 20x of magnification of the individually captured image of L3 group a. Qualitative analysis of the immunofluorescence staining for Collagen I and III showed a positive immunoreactivity in all experimental groups.

The muscle fibers expressing Collagens I and III were detected in the endomysium and perimysium area of the TA muscle. Compared to Collagen I, it could be seen that Collagen III is more active than Collagen I 7 days after cryolesion Fig.

Cryotherapy treatment did not change these results compared with the injured group. Micrographs are representative of muscle cross sections observed for control group of collagen I a and collage III b and for L7 groups collagen I c and for collagen III d.

The muscle fibers expressing collagen I and III were detected in the endomysium and perimysium area of the TA muscle of control group A and B-arrows.

Injury area are positive stained for both collagens 7 days post-lesion c and d — asterisks. Cryotherapy treatment did not alter the percentage of collagen immunoreactivity fibers in all periods.

These results provide new information about the effects of clinical-like cryotherapy on the molecular pathways involved in TA during muscle. They were characterized by a decreased in inflammatory process, however cryotherapy did not enhance muscle repair and collagen content.

The reduction in inflammatory processes could associated to attenuation of pain after muscle injury and could promote structural and functional restoration, which in turn facilitates rehabilitation 35 , Nevertheless, studies in humans are also necessary to examine this hypothesis, since the physiological significance of this reduction in inflammation, in the face of a lack of effect on repair must be clinically determined.

Although cryotherapy was hailed as advantageous in terms of reducing pain, swelling, degeneration and inflammation post-injury in sports medicine 3 , 15 , 16 , 17 , the results of studies comparing the effectiveness of cryotherapy on muscle regeneration are inconsistent and do not confirm this claim.

Schaser et al. Merrick et al. Despite the biological contribution from the effects of cryotherapy, those protocols used by Schaser et al. In addition, continuous cryotherapy lasting for several hours is associated with a certain risk of adverse effects, such as local skin injury 25 , Therefore, we only found three studies with results comparable to ours that used intermittent and clinical-like protocols related to those used in humans 4 , 22 , Data from the present study showed that cryotherapy did not alter the muscle-injured area and the expression of related factors for muscle regeneration Desmin and MyoD at 3, 7 and 14 days post-injury.

These results are interesting when compared with those of Oliveira and colleagues 22 , The absence of cryotherapeutic effects on muscle injury and markers for muscle regeneration in the present study could be also attributed to the period 3 days, 7 and 14 days after post-lesion of evaluation in comparison to those studies.

Interestingly, the negative effects of cryotherapy on muscle regeneration showed by Takagi et al. Some studies observed that macrophages are crucial in myoblast proliferation and differentiation for forming myotubes 7 , 8. Satellite cell activity could be also regulated by growth factors and cytokines secreted by neutrophils and macrophages, such as IGF, TNF-α and TGF-β 6 , 38 , According to Takagi et al.

The present study also observed that cryotherapy decreased TGF-β1 and IGF-1 expression, as well as the percentage of CD68 cells macrophages at 3 and 7 days post-injury.

Journal Muscle regeneration process Experimental Orthopaedics volume 3Article number: prkcess Cite Herbal alternative medicine article. Reteneration details. Satellite rwgeneration are tissue resident muscle stem cells required for postnatal skeletal muscle growth and repair through replacement of damaged myofibers. Muscle regeneration is coordinated through different mechanisms, which imply cell-cell and cell-matrix interactions as well as extracellular secreted factors. Cellular dynamics during muscle regeneration are highly complex. Adult Blood pressure management muscle reegneration a remarkable ability to regeneratipn. Regeneration of mature muscle fibers is dependent regenfration muscle stem cells Building muscular endurance satellite Vitamin A benefits. Although they are normally Muscle regeneration process a quiescent state, satellite regenerarion are rapidly activated after injury, and subsequently proliferate and differentiate to make new muscle fibers. Myogenesis is a highly orchestrated biological process and has been extensively studied, and therefore many parameters that can precisely evaluate regenerating events have been established. However, in some cases, it is necessary to evaluate the completion of regeneration rather than ongoing regeneration. In this study, we establish methods for assessing the myofiber maturation during muscle regeneration.

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What makes muscles grow? - Jeffrey Siegel

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