Publications

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin- proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

JTD Keywords: 3d bioengineered skeletal muscle tissues, Adrenal cortex hormones, Atroph, Colocalization, Corticosteroids, Dexamethasone, Glucocorticoid-receptor, Humans, Mechanisms, Models, biological, Mtor protein, human, Muscle contraction, Muscle fibers, skeletal, Muscle strength, Muscle, skeletal, Muscular diseases, Organ size, Phosphorylation, Proteasome endopeptidase complex, Proto-oncogene proteins c-akt, Receptors, glucocorticoid, Signal transduction, Skeletal-muscle, Steroid myopathy, Steroids, Supplementation, Taurin, Taurine, Tor serine-threonine kinases, Ubiquitin

Surface electromyography (sEMG) can be used to measure the electrical activity of the respiratory muscles. The possible applications of sEMG span from patients suffering from acute respiratory failure to patients receiving chronic home mechanical ventilation, to evaluate muscle function, titrate ventilatory support and guide treatment. However, sEMG is mainly used as a monitoring tool for research and its use in clinical practice is still limited-in part due to a lack of standardization and transparent reporting. During this round table meeting, recommendations on data acquisition, processing, interpretation, and potential clinical applications of respiratory sEMG were discussed. This paper informs the clinical researcher interested in respiratory muscle monitoring about the current state of the art on sEMG, knowledge gaps and potential future applications for patients with respiratory failure.

JTD Keywords: Acute respiratory failure, Artificial ventilation, Asthmatic-children, Breathing muscle, Clinical monitoring, Clinical practice, Clinical research, Consensus development, Data interpretation, Disease exacerbation, Drive, Electrode positioning, Electrode removal, Electromyography, Force, Home care, Human, Human diaphragm, Humans, Information processing, Inspiratory muscle training, Inspiratory muscles, Intensive care unit, Knowledge gap, Long term care, Mechanical ventilation, Medical procedures, Muscle contraction, Muscle fatigue, Muscle function, Muscle training, Muscle, skeletal, Muscle-activity, Noninvasive ventilation, Patient monitoring, Patient-ventilator asynchrony, Physiology, Prognosis, Quality of life, Reporting and data system, Respiratory failure, Respiratory muscles, Review, Severe exacerbations, Signal processing, Skeletal muscle, Standardization, Surface electromyography, Time factor

Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disease diagnosed in childhood. It is a progressive and wasting disease, characterized by a degeneration of skeletal and cardiac muscles caused by the lack of dystrophin protein. The absence of this crucial structural protein leads to sarcolemmal fragility, resulting in muscle fiber damage during contraction. Despite ongoing efforts, there is no cure available for DMD patients. One of the primary challenges is the limited efficacy of current preclinical tools, which fail in modeling the biological complexity of the disease. Human-based three-dimensional (3D) cell culture methods appear as a novel approach to accelerate preclinical research by enhancing the reproduction of pathophysiological processes in skeletal muscle. In this work, we developed a patient-derived functional 3D skeletal muscle model of DMD that reproduces the sarcolemmal damage found in the native DMD muscle. These bioengineered skeletal muscle tissues exhibit contractile functionality, as they responded to electrical pulse stimulation. Sustained contractile regimes induced the loss of myotube integrity, mirroring the pathological myotube breakdown inherent in DMD due to sarcolemmal instability. Moreover, damaged DMD tissues showed disease functional phenotypes, such as tetanic fatigue. We also evaluated the therapeutic effect of utrophin upregulator drug candidates on the functionality of the skeletal muscle tissues, thus providing deeper insight into the real impact of these treatments. Overall, our findings underscore the potential of bioengineered 3D skeletal muscle technology to advance DMD research and facilitate the development of novel therapies for DMD and related neuromuscular disorders.

JTD Keywords: 3d cell culture, disease modeling, drug testing, duchenne muscular dystrophy, sarcolemmal damage, skeletal muscle, 3d cell culture, Animal-models, Disease modeling, Dmso, Drug testing, Duchenne muscular dystrophy, Gene, Humans, Image, Mechanisms, Muscle fibers, skeletal, Muscle, skeletal, Muscular dystrophy, duchenne, Myocardium, Sarcolemmal damage, Skeletal muscle, Tissue engineering, Utrophin

Muscular dystrophies are a heterogeneous group of highly debilitating diseases that result in muscle atrophy and weakness. The lack of suitable cellular and animal models that reproduce specific aspects of their pathophysiology is one of the reasons why there are no curative treatments for these disorders. This highlights a considerable gap between current laboratory models and clinical practice. We strongly believe that organs-on-chip could help to fill this gap. Organs-on-chip, and in particular muscles-on-chip, are microfluidic devices that integrate functional skeletal muscle tissues. Biosensors in these systems allow monitoring of muscle homeostasis or drug responses in situ. This Perspective outlines the potential of organs-on-chip as advanced models for muscular dystrophies, as well as the current challenges and future opportunities for this technology.© 2023. Published by The Company of Biologists Ltd.

JTD Keywords: cell, tissue, Animals, Lab-on-a-chip devices, Muscle, skeletal, Muscular dystrophies, Skeletal-muscle

Abstract Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human platelet lysate reinforced with cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications.

JTD Keywords: 3d culture, generation, identification, image, manipulate, matrigel, mechanics, model, platelet lysate, scaffolds, skeletal muscle, tissue engineering, xeno-free, 3d culture, Animals, Extracellular matrix, Humans, Hydrogels, Muscle development, Muscle, skeletal, Platelet lysate, Platform, Skeletal muscle, Tissue engineering, Tissue scaffolds, Xeno-free

Three-dimensional engineering of skeletal muscle is becoming increasingly relevant for tissue engineering, disease modeling and bio-hybrid robotics, where flexible, versatile and multidisciplinary approaches for the evaluation of tissue differentiation, functionality and force measurement are required. This works presents a 3D-printed platform of bioengineered human skeletal muscle which can efficiently model the three-dimensional structure of native tissue, while providing information about force generation and contraction profiles. Proper differentiation and maturation of myocytes is demonstrated by the expression of key myo-proteins using immunocytochemistry and analyzed by confocal microscopy, and the functionality assessed via electrical stimulation and analysis of contraction kinetics. To validate the flexibility of this platform for complex tissue modeling, the bioengineered muscle is treated with tumor necrosis factor α to mimic the conditions of aging, which is supported by morphological and functional changes. Moreover, as a proof of concept, the effects of Argireline® Amplified peptide, a cosmetic ingredient that causes muscle relaxation, are evaluated in both healthy and aged tissue models. Therefore, the results demonstrate that this 3D-bioengineered human muscle platform could be used to assess morphological and functional changes in the aging process of muscular tissue with potential applications in biomedicine, cosmetics and bio-hybrid robotics.

JTD Keywords: 3d bioprinting, bio-actuator, drug testing, human skeletal muscle, muscle ageing, platform, tnf-alpha, 3d bioprinting, Aged, Aging, Bio-actuator, Bioprinting, Drug testing, Human skeletal muscle, Humans, Muscle ageing, Muscle, skeletal, Necrosis-factor-alpha, Pharmaceutical preparations, Tissue engineering

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.

JTD Keywords: 3d cell culture, hydrogel micropatterning, myotonic dystrophy, skeletal muscle, tissue engineering, 3d cell culture, Animals, Cell differentiation, Humans, Hydrogel micropatterning, Muscle fibers, skeletal, Muscle, skeletal, Myoblasts, Myotonic dystrophy, Skeletal muscle, Tissue engineering

Bioinspired hybrid soft robots that combine living and synthetic components are an emerging field in the development of advanced actuators and other robotic platforms (i.e., swimmers, crawlers, and walkers). The integration of biological components offers unique characteristics that artificial materials cannot precisely replicate, such as adaptability and response to external stimuli. Here, we present a skeletal muscle–based swimming biobot with a three-dimensional (3D)–printed serpentine spring skeleton that provides mechanical integrity and self-stimulation during the cell maturation process. The restoring force inherent to the spring system allows a dynamic skeleton compliance upon spontaneous muscle contraction, leading to a cyclic mechanical stimulation process that improves the muscle force output without external stimuli. Optimization of the 3D-printed skeletons is carried out by studying the geometrical stiffnesses of different designs via finite element analysis. Upon electrical actuation of the muscle tissue, two types of motion mechanisms are experimentally observed: directional swimming when the biobot is at the liquid-air interface and coasting motion when it is near the bottom surface. The integrated compliant skeleton provides both the mechanical self-stimulation and the required asymmetry for directional motion, displaying its maximum velocity at 5 hertz (800 micrometers per second, 3 body lengths per second). This skeletal muscle–based biohybrid swimmer attains speeds comparable with those of cardiac-based biohybrid robots and outperforms other muscle-based swimmers. The integration of serpentine-like structures in hybrid robotic systems allows self-stimulation processes that could lead to higher force outputs in current and future biomimetic robotic platforms. Copyright © 2021 The Authors, some rights reserved;

JTD Keywords: actuators, design, fabrication, mechanics, mems, myotubes, platform, tissue, 3d printers, Agricultural robots, Animals, Artificial organs, Biological components, Biomimetic materials, Biomimetic processes, Biomimetics, Cell line, Electrical actuation, Equipment design, Finite element analysis, Geometrical stiffness, Intelligent robots, Liquefied gases, Liquid-air interface, Mechanical integrity, Mechanical phenomena, Mechanical stimulation, Mice, Motion, Muscle, Muscle contractions, Muscle, skeletal, Phase interfaces, Printing, three-dimensional, Robotics, Serpentine, Smart materials, Springs (components), Swimming, Threedimensional (3-d), Tissue scaffolds

© 1964-2012 IEEE. Surface electromyography (sEMG) can be used for the evaluation of respiratory muscle activity. Recording sEMG involves the use of surface electrodes in a bipolar configuration. However, electrocardiographic (ECG) interference and electrode orientation represent considerable drawbacks to bipolar acquisition. As an alternative, concentric ring electrodes (CREs) can be used for sEMG acquisition and offer great potential for the evaluation of respiratory muscle activity due to their enhanced spatial resolution and simple placement protocol, which does not depend on muscle fiber orientation. The aim of this work was to analyze the performance of CREs during respiratory sEMG acquisitions. Respiratory muscle sEMG was applied to the diaphragm and sternocleidomastoid muscles using a bipolar and a CRE configuration. Thirty-two subjects underwent four inspiratory load spontaneous breathing tests which was repeated after interchanging the electrode positions. We calculated parameters such as (1) spectral power and (2) median frequency during inspiration, and power ratios of inspiratory sEMG without ECG in relation to (3) basal sEMG without ECG (Rins/noise), (4) basal sEMG with ECG (Rins/cardio) and (5) expiratory sEMG without ECG (Rins/exp). Spectral power, Rins/noise and Rins/cardio increased with the inspiratory load. Significantly higher values (p < 0.05) of Rins/cardio and significantly higher median frequencies were obtained for CREs. Rins/noise and Rins/exp were higher for the bipolar configuration only in diaphragm sEMG recordings, whereas no significant differences were found in the sternocleidomastoid recordings. Our results suggest that the evaluation of respiratory muscle activity by means of sEMG can benefit from the remarkably reduced influence of cardiac activity, the enhanced detection of the shift in frequency content and the axial isotropy of CREs which facilitates its placement.

JTD Keywords: atmospheric measurements, concentric ring electrodes, electrocardiography, electrodes, electromyography, laplacian potential, non-invasive respiratory monitoring, particle measurements, respiratory muscles, surface electromyography, Concentric ring electrodes, Diaphragm, Electrocardiography, Electrodes, Electromyography, Humans, Laplacian potential, Muscle, skeletal, Muscles, Non-invasive respiratory monitoring, Respiratory muscles, Surface electromyography