Publications

Biofabrication of three-dimensional (3D) cultures through the 3D Bioprinting technique opens new perspectives and applications of cell-laden hydrogels. However, to continue with the progress, new BioInks with specific properties must be carefully designed. In this study, we report the synthesis and 3D Bioprinting of an electroconductive BioInk made of gelatin/fibrinogen hydrogel, C2C12 mouse myoblast and 5% w/w of conductive poly (3,4-ethylenedioxythiophene) nanoparticles (PEDOT NPs). The influence of PEDOT NPs, incorporated in the cellladen BioInk, not only showed a positive effect in cells viability, differentiation and myotube functionalities, also allowed the printed constructs to behaved as BioCapacitors. Such devices were able to electrochemically store a significant amount of energy (0.5 mF/cm2), enough to self-stimulate as BioActuator, with typical contractions ranging from 27 to 38 mu N, during nearly 50 min. The biofabrication of 3D constructs with the proposed electroconductive BioInk could lead to new devices for tissue engineering, biohybrid robotics or bioelectronics.

JTD Keywords: 3d bioprinting, Animal, Animals, Bioactuator, Bioactuators, Biocapacitor, Biofabrication, Bioprinting, Biosensing techniques, C2c12 myoblasts, Cells, Chemistry, Electric conductivity, Electroconductive, Electroconductive bioink, Ethylenedioxythiophenes, Genetic procedures, Hydrogel, Hydrogels, Mice, Mouse, Pedot nps, Pedot nps,3d bioprinting,electroconductive bioink,bioactuator,biocapacito, Poly (3,4-ethylenedioxythiophene) nanoparticle, Printing, three-dimensional, Procedures, Skeletal-muscle,cytotoxicity,polymer, Synthesis (chemical), Three dimensional printing, Tissue engineering, Tissue scaffolds

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

The incorporation of exogenous lactate into cardiac tissues is a regenerative strategy that is rapidly gaining attention. In this work, two polymeric platforms were designed to achieve a sustained release of lactate, combining immediate and prolonged release profiles. Both platforms contained electrospun poly(lactic acid) (PLA) fibers and an alginate (Alg) hydrogel. In the first platform, named L/K(x)/Alg-PLA, lactate and proteinase K (x mg of enzyme per 1 g of PLA) were directly loaded into the Alg hydrogel, into which PLA fibers were assembled. In the second platform, L/Alg-K(x)/PLA, fibers were produced by electrospinning a proteinase K:PLA solution and, subsequently, assembled within the lactate-loaded hydrogel. After characterizing the chemical, morphological, and mechanical properties of the systems, as well as their cytotoxicity, the release profiles of the two platforms were determined considering different amounts of proteinase K (x = 5.2, 26, and 52 mg of proteinase K per 1 g of PLA), which is known to exhibit a broad cleavage activity. The profiles obtained using L/Alg-K(x)/PLA platforms with x = 26 and 52 were the closest to the criteria that must be met for cardiac tissue regeneration. Finally, the amount of lactate directly loaded in the Alg hydrogel for immediate release and the amount of protein in the electrospinning solution were adapted to achieve a constant lactate release of around 6 mM per day over 1 or 2 weeks. In the optimized bioplatform, in which 6 mM lactate was loaded in the hydrogel, the amount of fibers was increased by a factor of ×3, the amount of enzyme was adjusted to 40 mg per 1 g of PLA, and a daily lactate release of 5.9 ± 2.7 mM over a period of 11 days was achieved. Accordingly, the engineered device fully satisfied the characteristics and requirements for heart tissue regeneration.

JTD Keywords: biodegradable fibers, cardiac tissue regeneration, cell, drug-release, elastic-modulus, electrospinning, heart, nanoindentation, plasma treatment, proteinase, scaffold, stiffness, Alginate, Biodegradable fibers, Cardiac tissue regeneration, Electrospinning, Nanoindentation, Plasma treatment, Proteinase, Skeletal-muscle

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

Biohybrid robots, or bio-bots, integrate living and synthetic materials following a synergistic strategy to acquire some of the unique properties of biological organisms, like adaptability or bio-sensing, which are difficult to obtain exclusively using artificial materials. Skeletal muscle is one of the preferred candidates to power bio-bots, enabling a wide variety of movements from walking to swimming. Conductive nanocomposites, like gold nanoparticles or graphene, can provide benefits to muscle cells by improving the scaffolds' mechanical and conductive properties. Here, boron nitride nanotubes (BNNTs), with piezoelectric properties, are integrated in muscle-based bio-bots and an improvement in their force output and motion speed is demonstrated. A full characterization of the BNNTs is provided, and their piezoelectric behavior with piezometer and dynamometer measurements is confirmed. It is hypothesized that the improved performance is a result of an electric field generated by the nanocomposites due to stresses produced by the cells during differentiation. This hypothesis is backed with finite element simulations supporting that this stress can generate a non-zero electric field within the matrix. With this work, it is shown that the integration of nanocomposite into muscle-based bio-bots can improve their performance, paving the way toward stronger and faster bio-hybrid robots.

JTD Keywords: Bio-bots, Biohybrid robots, Biomaterials, Boron nitride nanotubes, Cells, Cytotoxicity, Differentiation, Myoblasts, Skeletal muscle tissue, Skeletal-muscle, Stimulation

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.

JTD Keywords: alpha-3 chain, binding, collagen vi related muscular dystrophy, decellularisation, decellularized matrices, deficiency, expression, extracellular matrix, fibroblasts, fibronectin, in vitro model, patient-derived ecms, skeletal-muscle, ullrich, Cell-derived matrices, Collagen, Collagen vi related muscular dystrophy, Decellularisation, Decellularization, Extracellular matrices, Extracellular matrix, Genes, In vitro model, In-vitro, In-vitro models, Matrix, Matrix model, Muscular dystrophy, Pathology, Patient-derived ecm, Patient-derived ecms, Pre-clinical

The cytoskeleton is a complex polymer network that regulates the structural stability of living cells. Although the cytoskeleton plays a key role in many important cell functions, the mechanisms that regulate its mechanical behaviour are poorly understood. Potential mechanisms include the entropic elasticity of cytoskeletal filaments, glassy-like inelastic rearrangements of cross-linking proteins and the activity of contractile molecular motors that sets the tensional stress (prestress) borne by the cytoskeleton filaments. The contribution of these mechanisms can be assessed by studying how cell mechanics depends on temperature. The aim of this work was to elucidate the effect of temperature on cell mechanics using atomic force microscopy. We measured the complex shear modulus (G*) of human alveolar epithelial cells over a wide frequency range (0.1-25.6 Hz) at different temperatures (13-37 degrees C). In addition, we probed cell prestress by mapping the contractile forces that cells exert on the substrate by means of traction microscopy. To assess the role of actomyosin contraction in the temperature-induced changes in G* and cell prestress, we inhibited the Rho kinase pathway of the myosin light chain phosphorylation with Y-27632. Our results show that with increasing temperature, cells become stiffer and more solid-like. Cell prestress also increases with temperature. Inhibiting actomyosin contraction attenuated the temperature dependence of G* and prestress. We conclude that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors.

JTD Keywords: Membrane Stress Failure, Frog Skeletal-Muscle, Extracellular-Matrix, Glass-Transition, Energy Landscape, Actin-Filaments, Living Cell, Single, Traction, Cytoskeleton