Anotherobstacleinmuscleregenerationisthemusculotendinous junction. This can be partly restored in absence of implanted cells by extracellular matrix-based platforms that have been shown to withstand half of the force of the contralateral site after complete resection in a mammalian model[80].Thenewlyformedmusclecellshaveshownbetter adherence to 3D polyurethane-based porous scaffolds with lowstiffnessandlargerroughnessvalues[84]. 3.2. Cell-Based Strategies. Muscle fiber regeneration is performed by cells and consequently cell-based strategies for regeneration have been pursued [83, 85]. The cell types utilizedfortreatingmusclelossmainlyincludemyoblasts,satellitecells(SCs),mesoangioblasts,pericytes,andmesenchymal stem cells (MSCs) [86–88]. The most well characterized musclestemcellisthesatellitecell(SC).SCsareabletocontributeextensivelytotheformationofnewmusclefibers[86, 89]. SCs transplanted into dystrophin-deficient mdx mice yieldedhighlyefficientregenerationofdystrophicmuscleand improvedmusclecontractilefunction[90].Unfortunately,in vitroexpansionofSCsresultsinsignificantreductionoftheir ability to produce myofibers in vivo [91] and consequently, obtainingasufficientlylargenumberoffreshSCsforclinical applicationisimpractical[92].Myoblastshavebeenusedfor reconstructingmuscletissuedefectswithavarietyofscaffolds [87, 93, 94]. They were shown to functionally integrate into the existing musculature of the host. Injection of a larger numberofmyoblastsintomusclesshowedpromisingresults for the treatment of dystrophin-deficient models [95]. Also MSCs could be involved in myotube formation through heterotypic cell fusion after myogenic gene activation [88]. Mesoangioblastsandpericyteshavebeenstudiedfortreating muscular dystrophy, which resulted in increasing the force [96]. They have also been utilized in tissue engineered hydrogelcarriers,withsomereportedsuccessforpromoting muscleregeneration[97]. Stem-cell-based therapies provide notable therapeutic benefitsonreversingmuscleatrophyandpromotingmuscle regeneration. Stem cell therapy (e.g., umbilical cord blood stemcelltransplantation)showedpositiveresultsfortreating Duchennemusculardystrophy[98].Afterapplicationofstem cells, an increase of dystrophin positive muscular fibers was found. Biopsies of calf muscle showed growing myoblasts cells and muscular tubes and an improvement in arms and legsduringphysicalexaminationwasreported. 3.3. Molecular Signaling Based Strategies. Beside cues from the ECM, also a diversity of stimulatory and inhibitory growthfactorssuchasIGF-1andTGF-ß1candriveendogenous skeletal muscle regeneration by activating and/or recruiting host stem cells [22]. They can be loaded on scaffolds for controlled delivery to the injured areas [72, 99]. Sustained delivery of VEGF, IGF-1, or SDF-1a was shown to enhance myogenesis and promote angiogenesis and muscle formation [73, 100–102]. Rapid release of hepatocyte growth factor (HGF) loaded on fibrin microthread scaffolds promoted remodeling of functional muscle tissue and enhanced the regeneration of skeletal muscle in mouse models [75]. Combination therapy of h-ADSCs and bFGF
hydrogels resulted in functional recovery, revascularization, andreinnervationinlaceratedmuscleswithminimalfibrosis [103]. Furthermore, PEDF peptide was reported to promote theregenerationofskeletalmuscles[104]. Research into the pathogenesis of sarcopenia as one of the most frequent muscular diseases has elucidated different molecular pathways. The most promising targets include BMP and myostatin [105]. Indeed, medication with human recombinant BMP-2/7 and antimyostatin can help toreducesarcopenicsymptoms[106].Cachexiaisaddressed with anamorelin, a ghrelin agonist, and selective androgen receptormodulatoraswellasanticytokines/myokines[107]. AnotherfactorinvolvedinmusclehealingseemstobeTGF𝛽. Increased TGF𝛽1 levels, which could be detected after the use of nonsteroidal anti-inflammatory drugs, helped to regeneratemuscletissue[108–110]. Spinal muscular atrophy arises from mutations in the survival motor neuron 1 (SMN1) gene, which often leads to thedeficiencyoftheubiquitousSMNprotein[111].Therefore, one of the most promising strategies is to increase the levels of full-length SMN [112]. Nusinersen is an antisense oligonucleotide drug developed for the treatment of spinal muscular atrophy (SMA), which has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) [113]. It can modulate the premRNA splicing of the survival motor neuron 2 gene and showed significant improvement of muscle function after treatment.Clinicaltrialsoninfantsshowedsignificantmean improvementsindevelopmentalmotormilestonesincluding sitting,walking,andmotorfunction[114]. 3.4.OtherDevelopingTechniques. Theeffectofheatstresson skeletalmuscleregenerationwasinvestigatedinexperimental rats [115]. Results showed that applying heat packs immediately after crush injury accelerated the degeneration process attheinjuredsite,facilitatedmigrationofmacrophages,proliferation,anddifferentiationofsatellitecells,andpromoted muscletissueregeneration. Low-level laser therapy (LLLT) has also been evaluated as a therapeutic approach for stimulating muscle repair and recoveryafterenduranceexercisetraininginrats[116].Other results from the rat model suggest that it could also be an optiontoreducefibrosisandmyonecrosistriggeredbybupivacaineandacceleratethemuscleregenerationprocess[117]. Aspossiblemechanisms,decreasedinflammationandmuscle creatinekinaselevelsarediscussed.ThecombinationofLLLT with platelet rich plasma (PRP) produced better results for promotingmuscleregenerationafterinjuriescomparedtothe isolateduseofLLLTorPRP[118]. The effect of neuromuscular electrical stimulation (NMES) on skeletal muscle regeneration was assessed in healthy subjects. It increased the proliferation of myogenic precursor cells (MPCs) and their fusion with mature myofibers, which improved the regenerative capacity of skeletal muscle [119]. The effect on models with muscle injuryorVMLneedstobefurtherinvestigate