Chapter III.9 - Muscle Tissue Engineering

While the effects of microgravity on inducing skeletal muscle atrophy have been extensively studied, the impacts of microgravity on myogenesis and its mechanisms remain unclear. In this study, we developed a microphysiological system of engineered muscle tissue (EMT) fabricated using a collagen / Matrigel composite hydrogel and murine skeletal myoblasts. This 3D EMT model allows non-invasive quantitative assessment of contractile function. After applying a 7-day differentiation protocol to induce myotube formation, the EMTs clearly exhibited sarcomerogenesis, myofilament formation, and synchronous twitch and tetanic contractions with electrical stimuli. Using this 3D EMT system, we investigated the effects of simulated microgravity at 10−3 G on myogenesis and contractile function utilizing a random positioning machine. EMTs cultured for 5 days in simulated microgravity exhibited significantly reduced contractile forces, myofiber size, and differential expression of muscle contractile, myogenesis regulatory, and mitochondrial biogenesis-related proteins. These results indicate simulated microgravity attenuates myogenesis, resulting in impaired muscle function.

Hierarchical multilayer assembly of an ordered nanofibrous scaffold via thermal fusion bonding

Tissue engineering methods that aim to mimic the hierarchical structure of skeletal muscle tissue have been widely developed due to utilities in various fields of biology, including regenerative medicine, food technology, and soft robotics. Most methods have aimed to reproduce the microscopical morphology of skeletal muscles, such as the orientation of myotubes and the sarcomere structure, and there is still a need to develop a method to reproduce the macroscopical morphology. Therefore, in this study, we aim to establish a method to reproduce the macroscopic morphology of skeletal muscle by constructing an engineered muscle tissue (EMT) by culturing embryonic chicken myoblast-like cells that are unidirectionally aligned in collagen hydrogels with micro-channels (i.e., MCCG). Whole mount fluorescent imaging of the EMT showed that the myotubes were unidirectionally aligned and that they were bundled in the collagen gel matrix. The myotubes contracted in response to periodic electrostimulations with a frequency range of 0.5–2.0 Hz, but not at 5.0 Hz. Compression tests of the EMT showed that the EMT had anisotropic elasticity. In addition, by measuring the relaxation moduli of the EMTs, an anisotropy of relaxation strengths was observed. The observed anisotropies could be attributed to differences in maturation and connectivity of myotubes in the directions perpendicular and parallel to the long axis of the micro-channels of the MCCG.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212nm vs. 284 ± 130nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Three-dimensional (3D) biomimetic systems hold great promise for the study of biological systems in vitro as well as for the development and testing of pharmaceuticals. Here, we test the hypothesis that an intact segment of lumbar rat spinal cord will form functional neuromuscular junctions (NMJs) with engineered, 3D muscle tissue, mimicking the partial development of the peripheral nervous system (PNS). Muscle tissues are grown on a 3D-printed polyethylene glycol (PEG) skeleton where deflection of the backbone due to muscle contraction causes the displacement of the pillar-like “feet.” We show that spinal cord explants extend a robust and complex arbor of motor neurons and glia in vitro. We then engineered a “spinobot” by innervating the muscle tissue with an intact segment of lumbar spinal cord that houses the hindlimb locomotor central pattern generator (CPG). Within 7 days of the spinal cord being introduced to the muscle tissue, functional neuromuscular junctions (NMJs) are formed, resulting in the development of an early PNS in vitro. The newly innervated muscles exhibit spontaneous contractions as measured by the displacement of pillars on the PEG skeleton. Upon chemical excitation, the spinal cord-muscle system initiated muscular twitches with a consistent frequency pattern. These sequences of contraction/relaxation suggest the action of a spinal CPG. Chemical inhibition with a blocker of neuronal glutamate receptors effectively blocked contractions. Overall, these data demonstrate that a rat spinal cord is capable of forming functional neuromuscular junctions ex vivo with an engineered muscle tissue at an ontogenetically similar timescale.

Microfluidic generation of helical micromotors for muscle tissue engineering

Muscle precursor cells (MPCs) are activated satellite cells capable of muscle fiber reconstruction. Therefore, autologous MPC transplantation is envisioned for the treatment of muscle diseases. However, the density of MPCs, as well as their proliferation and differentiation potential, gradually declines with age. The goals of this research were to genetically modify human MPCs (hMPCs) to overexpress the peroxisome proliferator-activated receptor γ coactivator (PGC-1α), a key regulator of exercise-mediated adaptation, and thereby to enhance early skeletal muscle formation and quality. We were able to confirm the sustained myogenic phenotype of the genetically modified hMPCs. While maintaining their viability and proliferation potential, PGC-1α-modified hMPCs showed an enhanced myofiber formation capacity in vitro. Engineered muscle tissues were harvested 1, 2, and 4 weeks after subcutaneous injection of cell–collagen suspensions, and histological analysis confirmed the earlier myotube formation in PGC-1α-modified samples, predominantly of slow-twitch myofibers. Increased contractile protein levels were detected by Western blot. In summary, by genetically modifying hMPCs to overexpress PGC-1α, we were able to promote early muscle fiber formation in vitro and in vivo, with an initial switch to slow-type myofibers. Therefore, overexpressing PGC-1α is a novel strategy to further enhance skeletal muscle tissue engineering.

The aim of skeletal muscle tissue engineering is to replace or repair skeletal muscle functions that have been injured or lost part of their functions. Skeletal muscle tissue engineering is an important strategy for muscle injury repair. The previous review on skeletal muscle tissue engineering was limited to the discussion of seed cells, biological materials and growth factors. In recent years, research results in this field have continued to emerge. This paper first briefly introduces the anatomy of skeletal muscle, and then combines the latest domestic and foreign study, from the basic problems of tissue engineering skeletal muscle construction to its vascularization, neuralization, etc., to review the current research status of skeletal muscle tissue engineering using three-dimensional (3D) printing, and look forward to its research prospects.

Achieving precise mechanical control in electrospun fibrous scaffolds remains a critical challenge for tissue engineering, where scaffold stiffness, strength, and extensibility must be tailored to diverse biological environments. Here, we establish a systematic framework for tuning the mechanical behavior of electrospun poly(ε-caprolactone) (PCL) fibers by integrating molecular-weight blending, polymer concentration control, fiber orientation, and environmental exposure within a single study. High-molecular-weight PCL (H-PCL) and blends with low-molecular-weight PCL (L-PCL) were electrospun to produce fibers with controlled diameters, morphologies, and orientations. Fiber alignment emerged as the dominant structural factor governing mechanical performance: oriented fibers exhibited substantially higher stiffness (~ 90–140 MPa) and tensile strength (up to ~ 100 MPa), while randomly deposited fibers showed markedly greater extensibility (up to ~ 1000%). Polymer concentration and resulting fiber diameter further modulated stiffness, with optimal mechanical performance observed at intermediate concentrations (~ 10–12% w/v). Molecular-weight blending provided an additional route to tailor fiber morphology and modulus, with oriented fibers reaching peak stiffness at ~ 50–60% H-PCL. Environmental exposure studies revealed that acidic treatments (formic and acetic acid solutions) reduce stiffness in a concentration- and temperature-dependent manner, whereas physiological soaking in phosphate-buffered saline (PBS, 37 °C) largely preserves scaffold integrity. Collectively, the electrospun scaffolds developed here span a broad mechanical window (~ 5–140 MPa). When positioned against literature-reported electrospun PCL scaffolds for cardiac, bone, and muscle tissue engineering, this range bridges multiple application-relevant stiffness regimes. These results provide a unified structure–property framework for designing mechanically tunable PCL fibrous scaffolds across diverse biomedical applications.

Skeletal muscle is a classical example of “structure determining function.” Successful strategies for clinical applications of tissue-engineered skeletal muscle must recapitulate the processes that muscle undergoes during either embryonic development or adult regeneration. As discussed in this chapter, current approaches to tissue engineering of skeletal muscle broadly utilize different aspects of these processes, e.g. the generation of multinucleate, elongated myotubes (nascent myofibres) surrounded by a connective tissue sheath. The former are generated by encouraging muscle precursor cell fusion, in which alignment is vital, so that surface topography, fibre scaffolds and mechanical loading are key processes. Connective tissue sheaths can be generated by encasing constructs in materials such as collagen and fibrin. Finally, functional connections need to be made to the skeletal system via tendons, to the neural system via neuromuscular junctions and to the vascular system. The chapter that follows reviews the current situation as regards these developments.

Myocardial tissue engineering ambitions to regenerate, repair or replace damaged cardiac muscle by combining cellular and engineering technologies. Several issues must be addressed before this approach may one day find clinical applications for cardiac disorders such as congenital diseases or ventricular dysfunction following myocardial infarction for example. The chance of the myocardial tissue engineering approach is nevertheless real. Indeed, on the one hand, several clinical studies have recently confirmed the positive effect of stem cell therapy in patients with heart failure. On the other hand, research from several laboratories have demonstrated over the past decade that engineered muscle tissues can be created and successfully applied in models of myocardial injury. Engineering a functional myocardial graft faces with many challenges, and various approaches have been investigated. In the current chapter, we focus our review on hydrogel-based engineered tissues for myocardial application. The literature on injectable and implantable hydrogel constructs is discussed and an overview of our own experience is presented. We emphasize important aspects on development of hydrogel constructs in particular the mechanical and electrical conditioning of the construct as well as smart hydrogels.

INTRODUCTIONIn this study, we investigated muscle regenerative ability in different age groups of experimental animals.METHODMuscle precursor cells (MPCs) were cultured from mice. In vitro analysis included cell growth assay, quantitative myotube formation. In vivo studies involved cells transfected or not transfected by adenovirus encoding Vascular Endothelial Growth Factor (VEGF). MPCs were injected subcutaneously into mice. After 2 and 4 weeks, the engineered muscle samples were harvested and analyzed.RESULTSThe young mice MPC growth rate and myotube formation were greater than mature mice and old mice MPC. Bioengineered muscle tissues were smaller and less contractile in old mice group. With expressing recombinant VEGF, bioengineered muscles in all age groups had a greater mass, increased neovascularization, and contractile function. Histological analysis of the in vivo engineered muscle tissue stained positive for muscle specific proteins.CONCLUSIONSThis study demonstrates the MPC regenerative possibilities in young and old mice, and augmentation of neovascularization increased muscle mass and strength of contractility of engineered muscle tissues. Funding NIH Ruth L. Kirschstein National Research Service Award Individual Fellowship (F31), the Novartis‐Stiftung, the Roche Research Foundation, GEBERT RÜF STIFTUNG, and CROWN Foundation.

The aetiology of pressure ulcers is poorly understood. The complexity of the problem, involving mechanical, biochemical, and physiological factors demands the need for simpler model systems that can be used to investigate the relative contribution of these factors, while controlling others. Therefore, an in vitro model system of engineered skeletal muscle tissue constructs was developed. With this model system, the relationship between compressive tissue straining and cell damage initiation was investigated under well-defined environmental conditions. Compression of the engineered muscle tissue constructs revealed that cell death occurs within 1-2 h at clinically relevant straining percentages and that higher strains led to earlier damage initiation. In addition, the uniform distribution of dead cells throughout the constructs suggested that sustained deformation of the cells was the principle cause of cell death. Therefore, it is hypothetised that sustained cell deformation is an additional mechanism that plays a role in the development of pressure ulcers.

Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch

Tissue engineering of skeletal muscle has been proposed as a potential regenerative treatment for extensive muscle damage. In this regard, the highly organized structure of skeletal muscles makes the alignment of cells especially indispensable in muscle tissue engineering. However, achieving the desired alignment continues to prove challenging, particularly in 3D engineered tissue constructs. In this study, a biomimetic approach for the generation of functional skeletal muscle fascicle‐like tissues by recapitulating 3D muscle‐like cellular and extracellular organization, is demonstrated. Anisotropic 3D alignment of muscle extracellular matrix (MEM) nanofibrils capable of providing a pro‐myogenic microenvironment by regulating the kinetics of fibrillogenesis in a stretchable elastomeric chip, is achieved. Reprogrammed muscle progenitor cells develop myofibers along the aligned MEM nanofibrils in a 3D configuration, culminating in the structural and functional maturation of skeletal muscle. The resultant 3D muscle fascicle‐like constructs support de novo muscle regeneration and induce functional restoration of injured muscles in animal models inflicted with volumetric muscle loss and congenital muscular dystrophy. This study not only highlights the fundamental roles of the muscle–mimetic structural guidance cues for 3D muscle tissue engineering, but also unveils the clinical potential of artificial muscle constructs in regenerative medicine.