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Spheroids exhibit enhanced cell-cell interactions that facilitate improved survival and mimic the physiological cellular environment in vivo. Cell spheroids have been successfully used as building blocks for engineered tissues, yet the viability of this approach with skeletal muscle spheroids is poorly understood, particularly when incorporated into three-dimensional (3D) constructs. Bioprinting is a promising strategy to recapitulate the hierarchical organization of native tissue that is fundamental to its function. However, the influence of bioprinting on skeletal muscle cell spheroids and their function are yet to be interrogated. Using C2C12 mouse myoblasts and primary bovine muscle stem cells (MuSCs), we characterized spheroid formation as a function of duration and cell seeding density. We then investigated the potential of skeletal muscle spheroids entrapped in alginate bioink as tissue building blocks for bioprinting myogenic tissue. Both C2C12 and primary bovine MuSCs formed spheroids of similar sizes and remained viable after bioprinting. Spheroids of both cell types fused into larger tissue clusters over time within alginate and exhibited tissue formation comparable to monodisperse cells. Compared to monodisperse cells in alginate gels, C2C12 spheroids exhibited greater MyHC expression after 2 weeks, while cells within bovine MuSC spheroids displayed increased cell spreading. Both monodisperse and MuSC spheroids exhibited increased expression of genes denoting mid- and late-stage myogenic differentiation. Together, these data suggest that skeletal muscle spheroids have the potential for generating myogenic tissue via 3D bioprinting and reveal areas of research that could enhance myogenesis and myogenic differentiation in future studies.

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of 'structural weakness', intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah-/- diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah-/- diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah-/- single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah-/- genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.

Key pointsThe amount of fibrotic material in dystrophic mouse muscles relates to contractile function, but not passive function. Collagen fibres in skeletal muscle are associated with increased passive muscle stiffness in fibrotic muscles. The alignment of collagen is independently associated with passive stiffness in dystrophic skeletal muscles. These outcomes demonstrate that collagen architecture rather than collagen content should be a target of anti-fibrotic therapies to treat muscle stiffness.AbstractFibrosis is prominent in many skeletal muscle pathologies including dystrophies, neurological disorders, cachexia, chronic kidney disease, sarcopenia and metabolic disorders. Fibrosis in muscle is associated with decreased contractile forces and increased passive stiffness that limits joint mobility leading to contractures. However, the assumption that more fibrotic material is directly related to decreased function has not held true. Here we utilize novel measurement of extracellular matrix (ECM) and collagen architecture to relate ECM form to muscle function. We used mdx mice, a model for Duchenne muscular dystrophy that becomes fibrotic, and wildtype mice. In this model, extensor digitorum longus (EDL) muscle was significantly stiffer, but with similar total collagen, while the soleus muscle did not change stiffness, but increased collagen. The stiffness of the EDL was associated with increased collagen crosslinking as determined by collagen solubility. Measurement of ECM alignment using polarized light microscopy showed a robust relationship between stiffness and alignment for wildtype muscle that broke down in mdx muscles. Direct visualization of large collagen fibres with second harmonic generation imaging revealed their relative abundance in stiff muscles. Collagen fibre alignment was linked to stiffness across all muscles investigated and the most significant factor in a multiple linear regression-based model of muscle stiffness from ECM parameters. This work establishes novel characteristics of skeletal muscle ECM architecture and provides evidence for a mechanical function of collagen fibres in muscle. This finding suggests that anti-fibrotic strategies to enhance muscle function and excessive stiffness should target large collagen fibres and their alignment rather than total collagen.

Purpose/Aim: Skeletal muscle tissue explants have been cultured and studied for nearly 100 years. These cultures, which retain complex tissue structure in an environment suited to precision manipulation and measurement, have led to seminal discoveries of the extrinsic and intrinsic mechanisms regulating contractility, metabolism and regeneration. This review discusses the two primary models of muscle explant: isolated myofiber and intact muscle.Materials and Methods: Relevant literature was reviewed and synthesized with a focus on the unique challenges and capabilities of each explant model.Results: Impactful past, current and future novel applications are discussed.Conclusions: Experiments using skeletal muscle explants have been integral to our understanding of the fundamentals of muscle physiology. As they are refined and adapted, they are poised to continue to inform the field for years to come.

Background/aimsCell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase.MethodsMice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function.ResultsUnder basal conditions, Mmp13-/- mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13-/- primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement.ConclusionThese results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

Cerebral palsy (CP) describes some upper motoneuron disorders due to non‐progressive disturbances occurring in the developing brain that cause progressive changes to muscle. While longer sarcomeres increase muscle stiffness in patients with CP compared to typically developing (TD) patients, changes in extracellular matrix (ECM) architecture can increase stiffness. Our goal was to investigate how changes in muscle and ECM architecture impact muscle stiffness, gait and joint function in CP. Gracilis and adductor longus biopsies were collected from children with CP undergoing tendon lengthening surgery for hamstring and hip adduction contractures, respectively. Gracilis biopsies were collected from TD patients undergoing anterior cruciate ligament reconstruction surgery with hamstring autograft. Muscle mechanical testing, two‐photon imaging and hydroxyproline assay were performed on biopsies. Corresponding data were compared to radiographic hip displacement in CP adductors (CPA), gait kinematics in CP hamstrings (CPH), and joint range of motion in CPA and CPH. We found at matched sarcomere lengths muscle stiffness and collagen architecture were similar between TD and CP hamstrings. However, CPH stiffness (R2 = 0.1973), collagen content (R2 = 0.5099) and cross‐linking (R2 = 0.3233) were correlated to decreased knee range of motion. Additionally, we observed collagen fibres within the muscle ECM increase alignment during muscular stretching. These data demonstrate that while ECM architecture is similar between TD and CP hamstrings, collagen fibres biomechanics are sensitive to muscle strain and may be altered at longer in vivo sarcomere lengths in CP muscle. Future studies could evaluate the impact of ECM architecture on TD and CP muscle stiffness across in vivo operating ranges. Key points: At matched sarcomere lengths, gracilis muscle mechanics and collagen architecture are similar in TD patients and patients with CP.In both TD and CP muscles, collagen fibres dynamically increase their alignment during muscle stretching.Aspects of muscle mechanics and collagen architecture are predictive of in vivo knee joint motion and radiographic hip displacement in patients with CP.Longer sarcomere lengths in CP muscle in vivo may alter collagen architecture and biomechanics to drive deficits in joint mobility and gait function. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of ‘structural weakness’, intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah−/− diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah−/− diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah−/− single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah−/− genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and homeostasis. However, dysregulation of these cells leads to fibro-fatty infiltration across various muscle diseases. FAPs are the key source of extracellular matrix (ECM) deposition in muscle, and disruption to this process leads to a pathological accumulation of ECM, known as fibrosis. The replacement of contractile tissue with fibrotic ECM functionally impairs the muscle and increases muscle stiffness. FAPs and fibrotic muscle form a progressively degenerative feedback loop where, as a muscle becomes fibrotic, it induces a fibrotic FAP phenotype leading to further development of fibrosis. In this review, we summarize FAPs' role in fibrosis in terms of their activation, heterogeneity, contributions to fibrotic degeneration, and role across musculoskeletal diseases. We also discuss current research on potential therapeutic avenues to attenuate fibrosis by targeting FAPs. [ABSTRACT FROM AUTHOR]

The muscle extracellular matrix (ECM) forms a complex network of collagens, proteoglycans, and other proteins that produce a favorable environment for muscle regeneration, protect the sarcolemma from contraction-induced damage, and provide a pathway for the lateral transmission of contractile force. In each of these functions, the structure and organization of the muscle ECM play an important role. Many aspects of collagen architecture, including collagen alignment, cross linking, and packing density affect the regenerative capacity, passive mechanical properties, and contractile force transmission pathways of skeletal muscle. The balance between fortifying the muscle ECM and maintaining ECM turnover and compliance is highly dependent on the integrated organization, or architecture, of the muscle matrix, especially related to collagen. While muscle ECM remodeling patterns in response to exercise and disease are similar, in that collagen synthesis can increase in both cases, one outcome leads to a stronger muscle and the other leads to fibrosis. In this review, we provide a comprehensive analysis of the architectural features of each layer of muscle ECM: epimysium, perimysium, and endomysium. Further, we detail the importance of muscle ECM architecture to biomechanical function in the context of exercise or fibrosis, including disease, injury, and aging. We describe how collagen architecture is linked to active and passive muscle biomechanics and which architectural features are acutely dynamic and adapt over time. Future studies should investigate the significance of collagen architecture in muscle stiffness, ECM turnover, and lateral force transmission in the context of health and fibrosis. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of 'structural weakness', intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah − / − diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah − / − diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah − / − single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah − / − genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background. [ABSTRACT FROM AUTHOR]

Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle, but in muscle pathologies FAPs the are main source of excess extracellular matrix (ECM) resulting in fibrosis. Fibrotic ECM has altered mechanical and architectural properties, but the feedback onto FAPs of stiffness or ECM properties is largely unknown. In this study, FAPs' sensitivity to their ECM substrate was assessed using collagen coated polyacrylamide to control substrate stiffness and collagen hydrogels to engineer concentration, crosslinking, fibril size, and alignment. FAPs on substrates of fibrotic stiffnesses had increased myofibroblast activation, depicted by αSMA expression, compared to substrates mimicking healthy muscle, which correlated strongly YAP nuclear localization. Surprisingly, fibrosis associated collagen crosslinking and larger fibril size inhibited myofibroblast activation, which was independent of YAP localization. Additionally, collagen crosslinking and larger fibril diameters were associated with decreased remodeling of the collagenous substrate as measured by second harmonic generation imaging. Inhibition of YAP activity through verteporfin reduced myofibroblast activation on stiff substrates but not substrates with altered architecture. This study is the first to demonstrate that fibrotic muscle stiffness can elicit FAP activation to myofibroblasts through YAP signaling. However, fibrotic collagen architecture actually inhibits myofibroblast activation through a YAP independent mechanism. These data expand knowledge of FAPs sensitivity to ECM and illuminate targets to block FAP's from driving progression of muscle fibrosis. [ABSTRACT FROM AUTHOR]

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

Introduction: Dysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice.Methods: Muscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice.Results: Muscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles.Discussion: We conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies. [ABSTRACT FROM AUTHOR]

Background Histological assessment of skeletal muscle tissue is commonly applied to many areas of skeletal muscle physiological research. Histological parameters including fiber distribution, fiber type, centrally nucleated fibers, and capillary density are all frequently quantified measures of skeletal muscle. These parameters reflect functional properties of muscle and undergo adaptation in many muscle diseases and injuries. While standard operating procedures have been developed to guide analysis of many of these parameters, the software to freely, efficiently, and consistently analyze them is not readily available. In order to provide this service to the muscle research community we developed an open source MATLAB script to analyze immunofluorescent muscle sections incorporating user controls for muscle histological analysis. Results The software consists of multiple functions designed to provide tools for the analysis selected. Initial segmentation and fiber filter functions segment the image and remove nonfiber elements based on user-defined parameters to create a fiber mask. Establishing parameters set by the user, the software outputs data on fiber size and type, centrally nucleated fibers, and other structures. These functions were evaluated on stained soleus muscle sections from 1-year-old wild-type and mdx mice, a model of Duchenne muscular dystrophy. In accordance with previously published data, fiber size was not different between groups, but mdx muscles had much higher fiber size variability. The mdx muscle had a significantly greater proportion of type I fibers, but type I fibers did not change in size relative to type II fibers. Centrally nucleated fibers were highly prevalent in mdx muscle and were significantly larger than peripherally nucleated fibers. Conclusions The MATLAB code described and provided along with this manuscript is designed for image processing of skeletal muscle immunofluorescent histological sections. The program allows for semi-automated fiber detection along with user correction. The output of the code provides data in accordance with established standards of practice. The results of the program have been validated using a small set of wild-type and mdx muscle sections. This program is the first freely available and open source image processing program designed to automate analysis of skeletal muscle histological sections. [ABSTRACT FROM AUTHOR]